U.S. patent application number 16/380963 was filed with the patent office on 2019-10-31 for multispecific polypeptide constructs having constrained cd3 binding and related methods and uses.
This patent application is currently assigned to Inhibrx, Inc.. The applicant listed for this patent is Inhibrx, Inc.. Invention is credited to Brendan P. ECKELMAN, Michael D. KAPLAN, John C. TIMMER, Katelyn M. WILLIS.
Application Number | 20190330366 16/380963 |
Document ID | / |
Family ID | 66248865 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190330366 |
Kind Code |
A1 |
ECKELMAN; Brendan P. ; et
al. |
October 31, 2019 |
MULTISPECIFIC POLYPEPTIDE CONSTRUCTS HAVING CONSTRAINED CD3 BINDING
AND RELATED METHODS AND USES
Abstract
The invention relates generally to multispecific polypeptides
having constrained CD3 binding. In some embodiments, components of
the multispecific polypeptides are connected by a non-cleavable
linker. Also provided are methods of making and using these
multispecific polypeptides in a variety of therapeutic, diagnostic
and prophylactic indications.
Inventors: |
ECKELMAN; Brendan P.; (La
Jolla, CA) ; KAPLAN; Michael D.; (La Jolla, CA)
; WILLIS; Katelyn M.; (La Jolla, CA) ; TIMMER;
John C.; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Inhibrx, Inc. |
La Jolla |
CA |
US |
|
|
Assignee: |
Inhibrx, Inc.
La Jolla
CA
|
Family ID: |
66248865 |
Appl. No.: |
16/380963 |
Filed: |
April 10, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62656331 |
Apr 11, 2018 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2809 20130101;
C07K 2317/522 20130101; C07K 2317/732 20130101; C07K 1/14 20130101;
C07K 2317/734 20130101; C07K 2317/622 20130101; C07K 2317/31
20130101; C07K 2317/526 20130101; C07K 2317/92 20130101; C07K
16/2878 20130101; A61P 35/00 20180101; C07K 16/30 20130101; C07K
2317/55 20130101; C07K 16/18 20130101; C07K 2317/66 20130101; C07K
2317/35 20130101; C07K 2317/624 20130101; C07K 2317/73 20130101;
C07K 2317/75 20130101; C07K 16/28 20130101; C07K 2317/567 20130101;
C07K 2317/64 20130101; C07K 16/2827 20130101; C07K 2317/569
20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61P 35/00 20060101 A61P035/00; C07K 1/14 20060101
C07K001/14 |
Claims
1. A multispecific polypeptide construct, the multispecific
polypeptide construct comprising a first component comprising an
immunoglobulin Fc region and a second component comprising a
CD3-binding region, wherein: the CD3 binding region is an anti-CD3
antibody or antigen binding fragment that is an Fv antibody
fragment comprising a variable heavy chain region (VH) and a
variable light chain region (VL); the Fc is a heterodimeric Fc
comprising a first Fc polypeptide and a second Fc polypeptide and
the VH and VL of the anti-CD3 antibody or antigen binding fragment
are linked to opposite polypeptides of the heterodimeric Fc; the
first and second components are coupled by a non-cleavable linker,
wherein the Fc region is positioned N-terminal to the CD3-binding
region; and the first component comprises a first antigen binding
domain and the second component comprises a second antigen binding
domain, wherein each of the antigen binding domains bind a tumor
associated antigen (TAA).
2. (canceled)
3. The multispecific polypeptide construct of claim 1, wherein the
first antigen binding domain is positioned amino-terminally
relative to the Fc region of the multispecific polypeptide
construct and the second antigen binding domain is positioned
carboxy-terminally relative to the CD3 binding region of the
multispecific polypeptide construct.
4. The multispecific polypeptide construct of claim 1, wherein the
multispecific polypeptide construct comprises in order, from
N-terminus to C-terminus: the first antigen binding domain that
binds to a tumor-associated antigen (TAA); the immunoglobulin Fc
region; the non-cleavable linker; the CD3 binding region, wherein
the CD3 binding region binds CD3 (CD3.epsilon.); and the second
antigen binding domain that binds a tumor-associated antigen
(TAA).
5. A multispecific polypeptide construct, the multispecific
polypeptide construct comprising a first component comprising an
immunoglobulin Fc region and a second component comprising a
CD3-binding region, wherein: the CD3 binding region is an anti-CD3
antibody or antigen binding fragment that is a disulfide-stabilized
Fv antibody fragment (dsFv) comprising a variable heavy chain (VH)
and a variable light chain (VL); the Fc is a heterodimeric Fc
comprising a first Fc polypeptide and a second Fc polypeptide and
the VH and VL of the anti-CD3 antibody or antigen binding fragment
are linked to opposite polypeptides of the heterodimeric Fc; the
first and second components are coupled by a non-cleavable linker,
wherein the Fc region is positioned N-terminal to the CD3-binding
region; and one or both of the first and second components
comprises an antigen binding domain that binds a tumor associated
antigen (TAA).
6. A multispecific polypeptide construct, the multispecific
polypeptide construct comprising a first component comprising an
immunoglobulin Fc region and a second component comprising a
CD3-binding region, wherein: the CD3 binding region is an anti-CD3
antibody or antigen binding fragment that is a an Fv antibody
fragment comprising a variable heavy chain (VH) and a variable
light chain (VL); the Fc is a heterodimeric Fc comprising a first
Fc polypeptide and a second Fc polypeptide and the VH and VL of the
anti-CD3 antibody or antigen binding fragment are linked to
opposite polypeptides of the heterodimeric Fc; the first and second
components are coupled by a non-cleavable linker, wherein the Fc
region is positioned N-terminal to the CD3-binding region; and one
or both of the first and second components comprises an antigen
binding domain that binds a tumor associated antigen (TAA), wherein
the antigen-binding domain is a single chain antibody fragment.
7. The multispecific polypeptide construct of claim 6, wherein the
single chain antibody fragment is a single domain antibody or is a
single chain variable fragment (scFv).
8. (canceled)
9. The multispecific polypeptide construct of claim 5, wherein the
multispecific polypeptide construct is selected from: (A) a
multispecific polypeptide construct that comprises in order, from
N-terminus to C-terminus: the first antigen binding domain that
binds to a tumor-associated antigen (TAA); the immunoglobulin Fc
region; the non-cleavable linker; the CD3 binding region, wherein
the CD3 binding region binds CD3 (CD3.epsilon.); and the second
antigen binding domain that binds a tumor-associated antigen (TAA);
or (B) a multispecific polypeptide construct that comprises in
order, from N-terminus to C-terminus: the immunoglobulin Fc region;
the non-cleavable linker; the CD3 binding region, wherein the CD3
binding region binds CD3 (CD3.epsilon.); and an antigen binding
domain that binds a tumor-associated antigen (TAA); or (C) a
multispecific polypeptide construct that comprises in order, from
N-terminus to C-terminus: the antigen binding domain that binds to
a tumor-associated antigen (TAA); the immunoglobulin Fc region; the
non-cleavable linker; and the CD3 binding region, wherein the CD3
binding region binds CD3 (CD3.epsilon.).
10. The multispecific polypeptide construct of claim 6, wherein the
multispecific polypeptide construct is selected from: (A) a
multispecific polypeptide construct that comprises in order, from
N-terminus to C-terminus: the first antigen binding domain that
binds to a tumor-associated antigen (TAA); the immunoglobulin Fc
region; the non-cleavable linker; the CD3 binding region, wherein
the CD3 binding region binds CD3 (CD3.epsilon.); and the second
antigen binding domain that binds a tumor-associated antigen (TAA);
or (B) a multispecific polypeptide construct that comprises in
order, from N-terminus to C-terminus: the immunoglobulin Fc region;
the non-cleavable linker; the CD3 binding region, wherein the CD3
binding region binds CD3 (CD3.epsilon.); and an antigen binding
domain that binds a tumor-associated antigen (TAA); or (C) a
multispecific polypeptide construct that comprises in order, from
N-terminus to C-terminus: the antigen binding domain that binds to
a tumor-associated antigen (TAA); the immunoglobulin Fc region; the
non-cleavable linker; and the CD3 binding region, wherein the CD3
binding region binds CD3 (CD3.epsilon.).
11-14. (canceled)
15. The multispecific polypeptide construct of claim 6, wherein the
heterodimeric Fc region comprises at least one modification
selected from among the following: a steric modification(s), a
knob-into-hole modification(s), a charge mutation(s) to increase
electrostatic complementarity of the polypeptides, a
modification(s) to alter the isoelectric point (pI variant), or
combinations thereof.
16-35. (canceled)
36. The multispecific polypeptide construct of claim 6, wherein the
linker is a polypeptide linker.
37. (canceled)
38. The multispecific polypeptide construct of claim 36, wherein
the linker is a polypeptide of from or from about 2 to 24 amino
acids.
39-43. (canceled)
44. The multispecific polypeptide construct of claim 6, wherein the
non-cleavable linker comprises: (GGS)n, wherein n is 1 to 10;
(GGGGS)n (SEQ ID NO: 173), wherein n is 1 to 10; or (GGGGGS)n (SEQ
ID NO:172), wherein n is 1 to 4.
45. (canceled)
46. (canceled)
47. The multispecific polypeptide construct of claim 6, wherein the
non-cleavable linker is or comprises an amino acid sequence
selected from among TABLE-US-00012 GGS; (SEQ ID NO: 149) GGGGS;
(SEQ ID NO: 135) GGGGGS; (SEQ ID NO: 10) (GGS).sub.2; (SEQ ID NO:
11) GGSGGSGGS; (SEQ ID NO: 12) GGSGGSGGSGGS; (SEQ ID NO: 13)
GGSGGSGGSGGSGGS; (SEQ ID NO: 119) GGGGGSGGGGGSGGGGGS; (SEQ ID NO:
147) GGSGGGGSGGGGSGGGGS; and (SEQ ID NO: 170) GGGGSGGGGSGGGGS.
48-56. (canceled)
57. The multispecific polypeptide construct of claim 6, wherein the
multispecific polypeptide construct comprises at least (i) a first
polypeptide comprising the first Fc polypeptide of the
heterodimeric Fc region, the linker and the VH domain of the
anti-CD3 antibody or antigen binding fragment thereof; and (ii) a
second polypeptide comprising the second Fc polypeptide of the
heterodimeric Fc region, the linker and the VL domain of the
anti-CD3 antibody or antigen binding fragment thereof, wherein one
or both of the first and second polypeptide comprise at least one
antigen-binding domain that binds to a tumor associated antigen
(TAA).
58. The multispecific polypeptide construct of claim 6, wherein the
VH of the anti-CD3 antibody or antigen-binding fragment is on the
same polypeptide as the at least one antigen-binding domain that
binds to a tumor associated antigen (TAA).
59. The multispecific polypeptide construct of claim 58, wherein
the polypeptide comprising the VL of the anti-CD3 antibody or
antigen-binding fragment does not contain the at least one
antigen-binding domain that binds to a tumor associated antigen
(TAA).
60. (canceled)
61. (canceled)
62. The multispecific polypeptide construct of claim 6, wherein
only one of the first and second polypeptide comprises the at least
one antigen-binding domain that binds a TAA.
63. The multispecific polypeptide construct of claim 6, wherein:
the at least one antigen binding domain is positioned
amino-terminally relative to the Fc region and/or is positioned
carboxy-terminally relative to the CD3 binding region of one of the
first or second polypeptide of the multispecific polypeptide
construct; or the at least one antigen binding domain comprises a
first and a second antigen binding domain and the first antigen
binding domain is positioned amino-terminally relative to the Fc
region of the multispecific polypeptide construct and the second
antigen binding domain is positioned carboxy-terminally relative to
the CD3 binding region of the multispecific polypeptide
construct.
64. (canceled)
65. The multispecific polypeptide construct of claim 1, wherein the
antigen binding domain, or independently each of the antigen
binding domains: comprises an extracellular domain or binding
fragment thereof of the native cognate binding partner of the TAA,
or a variant thereof that exhibits binding activity to the TAA; or
is an antibody or antigen-binding fragment thereof selected from
the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv
fragment, a scFv, a scAb, a dAb, a single domain heavy chain
antibody, and a single domain light chain antibody.
66. (canceled)
67. The multispecific polypeptide construct of claim 65, wherein
the antibody or antigen-binding fragment thereof is a Fv, a scFv, a
Fab, or a single domain antibody (sdAb).
68-77. (canceled)
78. The multispecific polypeptide construct of claim 6, wherein the
antigen binding domain, or independently each of the antigen
binding domains, binds to a tumor antigen selected from among
1-92-LFA-3, 5T4, Alpha-4 integrin, Alpha-V integrin, alpha4beta1
integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J
receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242,
CA9, CA19-9, (Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9,
CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38,
CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64,
CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125,
CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6,
CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet,
Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13,
CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1,
EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3,
EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8,
FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha
(FR.alpha.), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4,
GM-CSF, GM-CSFR, GP IIb/IIIa receptors, Gp130, GPIIB/IIIA, GPNMB,
GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS,
IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R,
IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbeta1, IL13,
IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27/IL27R (wsx1),
IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin
Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,
LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP,
Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF,
Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4,
NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta,
PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR,
RAAG12, RAGE, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2,
TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6,
TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFRS12A, TRAIL-R1,
TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR,
VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2,
VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.
79. The multispecific polypeptide construct of any of claim 6,
wherein the multispecific polypeptide construct comprises: at least
a first antigen binding domain and a second antigen binding domain,
wherein the first antigen binding domain and second antigen binding
domain bind to the same TAA; at least a first antigen binding
domain and a second antigen binding domain wherein the first
antigen binding domain and the second antigen binding domain binds
different epitopes of the same TAA; at least a first antigen
binding domain and a second antigen binding domain wherein the
first antigen binding domain and the second antigen binding domain
bind the same epitope of the same TAA; or at least a first antigen
binding domain and a second antigen binding domain wherein the
first antigen binding domain and the second antigen binding domain
bind a different TAA.
80-86. (canceled)
87. The multispecific polypeptide construct of claim 6, wherein the
Fv antibody fragment comprises a disulfide stabilized anti-CD3
binding Fv fragment (dsFv).
88-91. (canceled)
92. The multispecific polypeptide construct of claim 6, wherein the
multispecific polypeptide construct is conjugated to an agent
selected from among the following: a therapeutic agent, an
antineoplastic agent, a toxin or fragment thereof, a detectable
moiety, and a diagnostic agent.
93. (canceled)
94. (canceled)
95. A polynucleotide(s) encoding the mutispecific polypeptide
constructs of claim 6.
96-101. (canceled)
102. A vector, comprising the polynucleotide of claim 95.
103. (canceled)
104. (canceled)
105. A cell, comprising the polynucleotide or polynucleotides of
claim 95.
106-108. (canceled)
109. A method of producing a multispecific polypeptide construct,
the method comprising introducing into a cell the polynucleotide or
polynucleotides of claim 95 and culturing the cell under conditions
to produce the multispecific polypeptide construct.
110-113. (canceled)
114. A pharmaceutical composition comprising the multispecific
polypeptide construct of claim 6 and a pharmaceutically acceptable
carrier.
115. (canceled)
116. A method of stimulating or inducing an immune response, the
method comprising contacting a target cell and a T cell with the
multispecific polypeptide construct of claim 6, said target cell
expressing a tumor associated antigen recognized by the
multispecific polypeptide construct.
117-125. (canceled)
126. A method of treating a disease or condition in a subject, the
method comprising administering, to a subject in need thereof, a
therapeutically effective amount of the multispecific polypeptide
construct of claim 6.
127. The method of claim 126, wherein the disease or condition is a
tumor or a cancer.
128. (canceled)
129. The multispecific polypeptide construct of claim 1, wherein
the Fv antibody fragment comprises a disulfide stabilized anti-CD3
binding Fv fragment (dsFv).
130. The multispecific polypeptide construct of claim 5, wherein
the antigen binding domain, or independently each of the antigen
binding domains: comprises an extracellular domain or binding
fragment thereof of the native cognate binding partner of the TAA,
or a variant thereof that exhibits binding activity to the TAA; or
is an antibody or antigen-binding fragment thereof selected from
the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv
fragment, a scFv, a scAb, a dAb, a single domain heavy chain
antibody, and a single domain light chain antibody.
131. The multispecific polypeptide construct of claim 1, wherein
the non-cleavable linker is or comprises an amino acid sequence
selected from among GGS; GGGGS (SEQ ID NO: 149); GGGGGS (SEQ ID NO:
135); (GGS)2 (SEQ ID NO: 10); GGSGGSGGS (SEQ ID NO: 11);
GGSGGSGGSGGS (SEQ ID NO: 12); GGSGGSGGSGGSGGS (SEQ ID NO: 13);
GGGGGSGGGGGSGGGGGS (SEQ ID NO: 119); GGSGGGGSGGGGSGGGGS (SEQ ID NO:
147); and GGGGSGGGGSGGGGS (SEQ ID NO:170).
132. The multispecific polypeptide construct of claim 5, wherein
the non-cleavable linker is or comprises an amino acid sequence
selected from among GGS; GGGGS (SEQ ID NO: 149); GGGGGS (SEQ ID NO:
135); (GGS)2 (SEQ ID NO: 10); GGSGGSGGS (SEQ ID NO: 11);
GGSGGSGGSGGS (SEQ ID NO: 12); GGSGGSGGSGGSGGS (SEQ ID NO: 13);
GGGGGSGGGGGSGGGGGS (SEQ ID NO: 119); GGSGGGGSGGGGSGGGGS (SEQ ID NO:
147); and GGGGSGGGGSGGGGS (SEQ ID NO:170).
133. A polynucleotide(s) encoding the mutispecific polypeptide
constructs of claim 1.
134. A cell, comprising the polynucleotide or polynucleotides of
claim 133.
135. A method of producing a multispecific polypeptide construct,
the method comprising introducing into a cell the polynucleotide or
polynucleotides of claim 133 and culturing the cell under
conditions to produce the multispecific polypeptide construct.
136. A pharmaceutical composition comprising the multispecific
polypeptide construct of claim 1 and a pharmaceutically acceptable
carrier.
137. A method of stimulating or inducing an immune response, the
method comprising contacting a target cell and a T cell with the
multispecific polypeptide construct of claim 1, said target cell
expressing a tumor associated antigen recognized by the
multispecific polypeptide construct.
138. A method of treating a disease or condition in a subject, the
method comprising administering, to a subject in need thereof, a
therapeutically effective amount of the multispecific polypeptide
construct-of claim 1.
139. A polynucleotide(s) encoding the mutispecific polypeptide
constructs of claim 5.
140. A cell, comprising the polynucleotide or polynucleotides of
claim 139.
141. A method of producing a multispecific polypeptide construct,
the method comprising introducing into a cell the polynucleotide or
polynucleotides of claim 139 and culturing the cell under
conditions to produce the multispecific polypeptide construct.
142. A pharmaceutical composition comprising the multispecific
polypeptide construct of claim 5 and a pharmaceutically acceptable
carrier.
143. A method of stimulating or inducing an immune response, the
method comprising contacting a target cell and a T cell with the
multispecific polypeptide construct of claim 5, said target cell
expressing a tumor associated antigen recognized by the
multispecific polypeptide construct.
144. A method of treating a disease or condition in a subject, the
method comprising administering, to a subject in need thereof, a
therapeutically effective amount of the multispecific polypeptide
construct-of claim 5.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application 62/656,331, filed Apr. 11, 2018, entitled
"MULTISPECIFIC POLYPEPTIDE CONSTRUCTS HAVING CONSTRAINED CD3
BINDING AND RELATED METHODS AND USES," the contents of which are
incorporated by reference in their entirety for all purposes.
INCORPORATION BY REFERENCE OF SEQUENCE 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 744952000200SeqList.TXT, created Apr. 10, 2019 which
is 212 kilobytes in size. The information in the electronic format
of the Sequence Listing is incorporated by reference in its
entirety.
FIELD
[0003] The invention relates generally to multispecific
polypeptides having constrained CD3 binding. In some embodiments,
components of the multispecific polypeptides are connected by a
non-cleavable linker. Also provided are methods of making and using
these multispecific polypeptides in a variety of therapeutic,
diagnostic and prophylactic indications.
BACKGROUND
[0004] Therapeutic antibodies that cause target cell depletion
generally rely on effector functions mediated via interaction with
Fc-gamma-receptors (Fc.gamma.Rs) and complement proteins. Effector
cells expressing Fc.gamma.Rs are predominately those of the innate
immune system. T-cells are not direct effector cells involved in
antibody mediated target cell depletion.
[0005] CD3 (Cluster of Differentiation 3) T-cell co-receptor is a
multimeric protein composed of four distinct polypeptide chains,
referred to as the .epsilon., .gamma., .delta., and .zeta. chains.
The CD3 complex serves as the signaling module of the T cell
receptor that associates non-covalently with the antigen-binding
a/b chains of T cell receptor (TCR).
[0006] Because direct engagement of CD3 results in T-cell
activation, it is a desirable target for a variety of therapeutic
and/or diagnostic indications. Accordingly, there exists a need for
antibodies and therapeutics that target the CD3/TCR pathway.
SUMMARY
[0007] The present disclosure provides multispecific polypeptide
constructs that exhibit constrained CD3 binding. In some
embodiments, the multispecific polypeptide construct is composed of
a first component comprising an immunoglobulin Fc region and a
second component comprising a CD3-binding region, wherein the first
and second components are coupled by a linker, such as a
non-cleavable linker, wherein the Fc region is positioned
N-terminal to the CD3-binding region; and one or both of the first
and second components comprises an antigen binding domain that
binds a tumor associated antigen (TAA). In some embodiments, the
CD3-binding region binds CD3 (CD3.epsilon.). In some embodiments,
the antigen binding domain is positioned amino-terminally relative
to the Fc region and/or carboxy-terminally relative to the CD3
binding region of the multispecific polypeptide construct. In some
embodiments, the first component comprises a first antigen binding
domain and the second component comprises a second antigen binding
domain, wherein each of the antigen binding domains bind a tumor
associated antigen (TAA). In some cases, the first antigen binding
domain is positioned at the amino terminus of the multispecific
construct and the second antigen binding domain is positioned at
the carboxy terminus of the multispecific construct. In some
embodiments, the first antigen binding domain is positioned
amino-terminally relative to the Fc region and/or
carboxy-terminally relative to the CD3 binding region of the
multispecific polypeptide construct. In particular embodiments of
provided multispecific polypeptide constructs, at least one antigen
binding domain is positioned carboxy-terminally relative to the CD3
binding region of the multispecific polypeptide construct.
[0008] Provided herein is a multispecific polypeptide construct,
wherein the multispecific construct comprises in order, from
N-terminus to C-terminus: a first antigen binding domain that binds
to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a
linker, such as a non-cleavable linker, a CD3 binding region that
binds CD3 (CD3.epsilon.); and a second antigen binding domain that
binds a tumor-associated antigen (TAA). Also provided is a
multispecific polypeptide construct, wherein the multispecific
construct comprises in order, from N-terminus to C-terminus: an
immunoglobulin Fc region; a linker, such as a non-cleavable linker;
a CD3 binding region that binds CD3 (CD3.epsilon.); and an antigen
binding domain that binds a tumor-associated antigen (TAA).
Provided is a multispecific polypeptide construct, wherein the
multispecific construct comprises in order, from N-terminus to
C-terminus: an antigen binding domain that binds to a
tumor-associated antigen (TAA); an immunoglobulin Fc region; a
linker, such as a non-cleavable linker; and a CD3 binding region
that binds CD3 (CD3.epsilon.).
[0009] In some of any of the provided embodiments, the linker is a
non-cleavable linker. In some embodiments, the linker is a linker
that does not contain a substrate recognition site specific to
cleavage by a protease.
[0010] In some of any of the provided embodiments, the positioning
of the Fc region N-terminal to the CD3 binding region reduces or
prevents the ability of the CD3 binding region to bind CD3. In some
embodiments, the first component (component #1) and the second
component (component #2) of the multispecific polypeptide
constructs are linked and binding to CD3 is disallowed, unless the
antigen binding domain(s) is bound to its cognate antigen. In some
embodiments, component #1 contains at least one antigen binding
domain and an Fc region. In some embodiments, component #2 contains
at least a CD3 binding region domain and an antigen binding domain,
the former of which is capable of binding CD3 (when the
multispecific construct is bound to antigen recognized by the
antigen binding domain or domains of component #1 or component #2).
Thus, linkage of the CD3 binding region to the Fc region as
described ensures that the multispecific polypeptide constructs do
not bind or otherwise engage CD3 unless the antigen binding
domain(s) is bound to its cognate antigen. This is advantageous as
it prevents systemic binding of the CD3 binding region to T-cells
and instead focuses the CD3 binding region's ability to bind to
site of antigen expression. This is beneficial as it diminishes or
eliminates a major binding sink of peripheral T-cells, potentially
allowing more favorable distribution and localization at site of
antigen expression, e.g., tumor cells or the tumor
microenvironment.
[0011] When the antigen binding domain(s) is bound to its cognate
antigen, the multispecific polypeptide construct, via component #2,
is capable of forming an immune synapse between an
antigen-expressing cell and a T-cell. This co-engagement mediates
antigen dependent T-cell activation, cytotoxicity, cytokine
release, degranulation and proliferation. In some embodiments, the
multispecific polypeptide constructs are capable of interacting
with Fc.gamma.Rs and mediating innate immune effector functions,
for example antibody dependent cellular cytotoxicity (ADCC) and
antibody dependent cellular phagocytosis (ADCP). In some
embodiments, the multispecific polypeptide constructs are capable
of interacting with complement proteins, namely C1q, and mediating
complement-dependent cytotoxicity.
[0012] In some embodiments, the cognate antigen recognized by the
antigen binding domain(s) of a provided multispecific polypeptide
construct is a tumor associated antigen (TAA).
[0013] Thus, among the provided embodiments, the multispecific
polypeptide construct is composed of a first component comprising
an immunoglobulin Fc region and a second component comprising a
CD3-binding region, wherein the first and second components are
coupled by a linker, such as a non-cleavable linker, wherein the Fc
region is positioned N-terminal to the CD3-binding region; and one
or both of the first and second components comprises an antigen
binding domain that binds a tumor associated antigen (TAA). In some
embodiments, the CD3-binding region binds CD3 (CD3.epsilon.). In
some embodiments, the antigen binding domain is positioned
amino-terminally relative to the Fc region and/or
carboxy-terminally relative to the CD3 binding region of the
multispecific polypeptide construct. In some embodiments, the first
component comprises a first antigen binding domain and the second
component comprises a second antigen binding domain, wherein each
of the antigen binding domains bind a tumor associated antigen
(TAA). In some cases, the first antigen binding domain is
positioned at the amino terminus of the multispecific construct and
the second antigen binding domain is positioned at the carboxy
terminus of the multispecific construct. In some embodiments, the
first antigen binding domain is positioned amino-terminally
relative to the Fc region and/or carboxy-terminally relative to the
CD3 binding region of the multispecific polypeptide construct. In
particular embodiments of provided multispecific polypeptide
constructs, at least one antigen binding domain is positioned
carboxy-terminally relative to the CD3 binding region of the
multispecific polypeptide construct.
[0014] In some embodiments, the CD3 binding region is an antibody
or an antigen binding fragment. In particular embodiments, the
antibody or antigen binding fragment is a two chain polypeptide
containing a variable heavy (VH) and a variable light (VL) chain.
In some embodiments, the antibody or antigen-binding fragment is an
Fv. In particular embodiments, the Fv is a disulfide-stabilized Fv
(dsFv) containing an interchain disulfide bond between the VH and
VL chains.
[0015] Provided herein is a multispecific polypeptide construct
comprising a first component comprising an immunoglobulin Fc region
and a second component comprising a CD3-binding region, wherein the
CD3 binding region is an anti-CD3 antibody or antigen binding
fragment that is an Fv antibody fragment comprising a variable
heavy chain region (VH) and a variable light chain region (VL); the
Fc is a heterodimeric Fc comprising a first Fc polypeptide and a
second Fc polypeptide and the VH and VL of the anti-CD3 antibody or
antigen binding fragment are linked to opposite polypeptides of the
heterodimeric Fc; the first and second components are coupled by a
non-cleavable linker, wherein the Fc region is positioned
N-terminal to the CD3-binding region; and the first component
comprises a first antigen binding domain and the second component
comprises a second antigen binding domain, wherein each of the
antigen binding domains bind a tumor associated antigen (TAA). In
some embodiments, the CD3-binding region binds CD3 (CD3.epsilon.).
In some embodiments, the first antigen binding domain is positioned
amino-terminally relative to the Fc region of the multispecific
construct and the second antigen binding domain is positioned
carboxy-terminally relative to the CD3 binding region of the
multispecific construct. In some embodiments, the multispecific
construct comprises in order, from N-terminus to C-terminus: the
first antigen binding domain that binds to a tumor-associated
antigen (TAA); the immunoglobulin Fc region; the non-cleavable
linker; the CD3 binding region, wherein the CD3 binding region
binds CD3 (CD3.epsilon.); and the second antigen binding domain
that binds a tumor-associated antigen (TAA).
[0016] Provided herein is a multispecific polypeptide construct
comprising a first component comprising an immunoglobulin Fc region
and a second component comprising a CD3-binding region, wherein:
the CD3 binding region is an anti-CD3 antibody or antigen binding
fragment that is a disulfide-stabilized Fv antibody fragment (dsFv)
comprising a variable heavy chain (VH) and a variable light chain
(VL); the Fc is a heterodimeric Fc comprising a first Fc
polypeptide and a second Fc polypeptide and the VH and VL of the
anti-CD3 antibody or antigen binding fragment are linked to
opposite polypeptides of the heterodimeric Fc; the first and second
components are coupled by a non-cleavable linker, wherein the Fc
region is positioned N-terminal to the CD3-binding region; and one
or both of the first and second components comprises an antigen
binding domain that binds a tumor associated antigen (TAA). In
particular embodiments of provided multispecific polypeptide
constructs, at least one antigen binding domain is positioned
carboxy-terminally relative to the CD3 binding region of the
multispecific polypeptide construct. In some embodiments, the
CD3-binding region binds CD3 (CD3.epsilon.).
[0017] Provided herein is a multispecific polypeptide construct
comprising a first component comprising an immunoglobulin Fc region
and a second component comprising a CD3-binding region, wherein:
the CD3 binding region is an anti-CD3 antibody or antigen binding
fragment that is a an Fv antibody fragment comprising a variable
heavy chain (VH) and a variable light chain (VL); the Fc is a
heterodimeric Fc comprising a first Fc polypeptide and a second Fc
polypeptide and the VH and VL of the anti-CD3 antibody or antigen
binding fragment are linked to opposite polypeptides of the
heterodimeric Fc; the first and second components are coupled by a
non-cleavable linker, wherein the Fc region is positioned
N-terminal to the CD3-binding region; and one or both of the first
and second components comprises an antigen binding domain that
binds a tumor associated antigen (TAA), wherein the antigen-binding
domain is a single chain antibody fragment, such as an sdAb or an
scFv. In particular embodiments of provided multispecific
polypeptide constructs, at least one antigen binding domain is
positioned carboxy-terminally relative to the CD3 binding region of
the multispecific polypeptide construct. In some embodiments, the
CD3-binding region binds CD3 (CD3.epsilon.).
[0018] In embodiments provided herein, the multispecific construct
comprises in order, from N-terminus to C-terminus: the first
antigen binding domain that binds to a tumor-associated antigen
(TAA); the immunoglobulin Fc region; the non-cleavable linker; the
CD3 binding region, wherein the CD3 binding region binds CD3
(CD3.epsilon.); and the second antigen binding domain that binds a
tumor-associated antigen (TAA).
[0019] In embodiments provided herein, the multispecific construct
comprises in order, from N-terminus to C-terminus: the
immunoglobulin Fc region; the non-cleavable linker; the CD3 binding
region, wherein the CD3 binding region binds CD3 (CD3.epsilon.);
and an antigen binding domain that binds a tumor-associated antigen
(TAA).
[0020] In embodiments provided herein, the multispecific construct
comprises in order, from N-terminus to C-terminus: the antigen
binding domain that binds to a tumor-associated antigen (TAA); the
immunoglobulin Fc region; the non-cleavable linker; and the CD3
binding region, wherein the CD3 binding region binds CD3
(CD3.epsilon.).
[0021] Provided herein is a multispecific polypeptide construct,
wherein the multispecific construct comprises in order, from
N-terminus to C-terminus: a first antigen binding domain that binds
to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a
linker, such as a non-cleavable linker; a CD3 binding region that
binds CD3 (CD3.epsilon.); and a second antigen binding domain that
binds a tumor-associated antigen (TAA). Also provided is a
multispecific polypeptide construct, wherein the multispecific
construct comprises in order, from N-terminus to C-terminus: an
immunoglobulin Fc region; a linker, such as a non-cleavable linker;
a CD3 binding region that binds CD3 (CD3.epsilon.); and an antigen
binding domain that binds a tumor-associated antigen (TAA).
Provided is a multispecific polypeptide construct, wherein the
multispecific construct comprises in order, from N-terminus to
C-terminus: an antigen binding domain that binds to a
tumor-associated antigen (TAA); an immunoglobulin Fc region; a
linker, such as a non-cleavable linker; and a CD3 binding region
that binds CD3 (CD3.epsilon.).
[0022] In some aspects, the antigen binding domain, or
independently each of the antigen binding domains, is selected from
an antibody or antigen binding fragment, a natural cognate binding
partner, an Anticalin (engineered lipocalin), a Darpin, a Fynomer,
a Centyrin (engineered fibroneticin III domain), a cystine-knot
domain, an Affilin, an Affibody, or an engineered CH3 domain. In
some embodiments, the natural cognate binding partner comprises an
extracellular domain or binding fragment thereof of the native
cognate binding partner of the TAA, or a variant thereof that
exhibits binding activity to the TAA.
[0023] In some embodiments, the antigen-binding domain(s) includes
one or more copies of an antibody or an antigen-binding fragment
thereof. In some embodiments, the antigen-binding domain(s)
includes one or more copies of an antibody or an antigen-binding
fragment thereof selected from the group consisting of a Fab
fragment, a F(ab').sub.2 fragment, an Fv fragment, a scFv, a scAb,
a dAb, a single domain heavy chain antibody, and a single domain
light chain antibody. In some embodiments, the antigen-binding
domain(s) include one or more copies of one or more single domain
antibody (sdAb) fragments, for example V.sub.HH, V.sub.NAR,
engineered V.sub.H or V.sub.K domains. V.sub.HHs can be generated
from camelid heavy chain only antibodies. V.sub.NARs can be
generated from cartilaginous fish heavy chain only antibodies.
Various methods have been implemented to generate monomeric sdAbs
from conventionally heterodimeric V.sub.H and V.sub.K domains,
including interface engineering and selection of specific germline
families.
[0024] In some embodiments, the one or more antigen binding domains
independently bind an antigen that is a tumor associated antigen
(TAA). In some examples, the antigen binding domain, or
independently each of the antigen binding domains, binds to a tumor
antigen selected from among 1-92-LFA-3, 5T4, Alpha-4 integrin,
Alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2,
Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA,
C5 complement, C-242, CA9, CA19-9, (Lewis a), Carbonic anhydrase 9,
CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27,
CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47,
CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95,
CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166,
CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3,
CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF,
CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4,
DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin B receptor
(ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP,
FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor
alpha (FRa), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4,
GM-CSF, GM-CSFR, GP IIb/IIIa receptors, Gp130, GPIIB/IIIA, GPNMB,
GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS,
IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R,
IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbeta1, IL13,
IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27/IL27R (wsx1),
IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin
Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,
LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP,
Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF,
Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4,
NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta,
PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR,
RAAG12, RAGE, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2,
TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6,
TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFRS12A, TRAIL-R1,
TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR,
VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2,
VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.
[0025] In some embodiments, the Fc region is a homodimeric Fc
region. In some embodiments, the immunoglobulin Fc region of the
first component is an IgG isotype selected from the group
consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4
subclass. In some examples, the Fc region is an Fc region of a
human IgG1, a human IgG2, a human IgG3, or a human IgG4, or is an
immunologically active fragment thereof. In some embodiments, the
Fc region comprises a polypeptide comprises the amino acid sequence
set forth in SEQ ID NO: 1 or a sequence of amino acids that has at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence
identity to SEQ ID NO:1. In some cases, the Fc region comprises a
polypeptide comprises the amino acid sequence set forth in SEQ ID
NO: 2 or a sequence of amino acids that has at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2.
In some of any such embodiments, the Fc region comprises a
polypeptide comprises the amino acid sequence set forth in SEQ ID
NO: 4 or a sequence of amino acids that has at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:4.
In some examples, the Fc region comprises a polypeptide comprises
the amino acid sequence set forth in SEQ ID NO: 5 or a sequence of
amino acids that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% sequence identity to SEQ ID NO:5. In some examples,
the Fc region comprises a polypeptide comprises the amino acid
sequence set forth in SEQ ID NO: 6 or a sequence of amino acids
that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
sequence identity to SEQ ID NO:6.
[0026] In some embodiments, the immunoglobulin Fc region is a
polypeptide comprising an amino acid sequence that is derived from
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 1-6.
[0027] In some embodiments, the immunoglobulin Fc region is a
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 1-6. In some embodiments, the
immunoglobulin Fc region is a polypeptide comprising an amino acid
sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 1-6.
[0028] In some embodiments, the Fc region is a heterodimeric Fc
region.
[0029] In some embodiments, the Fc region is a heterodimer
containing a first Fc polypeptide and a second Fc polypeptide
wherein one or both of the first and second Fc polypeptides of the
heterodimeric Fc region are a variant Fc polypeptide comprising at
least one modification to induce heterodimerization compared to an
Fc region of human IgG1, human IgG2 or human IgG4. In some
embodiments, the at least one modification is in or compared to an
Fc region of human IgG1. In some embodiments, the at least one
modification is in or compared to the Fc polypeptide set forth in
SEQ ID NO:1 or an immunologically active fragment thereof. In some
cases, one or both Fc polypeptides of the heterodimeric Fc region
comprises at least one modification to induce heterodimerization
compared to a polypeptide of a homodimeric Fc region, optionally
compared to the Fc polypeptide set forth in SEQ ID NO:1 or an
immunologically active fragment thereof. In some embodiments, each
of the Fc polypeptides of the heterodimeric Fc independently
comprise at least one amino acid modification. In some cases, the
at least one modification is selected from a steric
modification(s), a knob-into-hole modification(s), a charge
mutation(s) to increase electrostatic complementarity of the
polypeptides, a modification(s) to alter the isoelectric point (p1
variant), or combinations thereof.
[0030] In some examples, the amino acid modification is a charge
mutation to increase electrostatic complementarity of the
polypeptides. In some embodiments, the first and/or second Fc
polypeptides comprise a modification in complementary positions,
wherein the modification is replacement with an amino acid having
an opposite charge to the complementary amino acid of the other
polypeptide. In some embodiments, the first or second polypeptide
comprise a modification in complementary positions, wherein the
modification is replacement with an amino acid having an opposite
charge to the complementary amino acid of the other polypeptide. In
some embodiments, at least the first or second Fc polypeptides each
comprise a modification in complementary positions, wherein the
modification is replacement with an amino acid having an opposite
charge to the complementary amino acid of the other polypeptide. In
some embodiments, the first and second Fc polypeptides each
comprise a modification in complementary positions, wherein the
modification is replacement with an amino acid having an opposite
charge to the complementary amino acid of the other
polypeptide.
[0031] In some examples, the amino acid modification is a
knob-into-hole modification.
[0032] In some embodiments, the first Fc polypeptide of the
heterodimeric Fc comprises the modification selected from among
Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof and the
second Fc polypeptide of the heterodimeric Fc comprises the
modification T366W. In some cases, the first and second Fc
polypeptides further comprise a modification of a non-cysteine
residue to a cysteine residue, wherein the modification of the
first polypeptide is at one of a position Ser354 and Y349 and the
modification of the second Fc polypeptide is at the other of the
position Ser354 and Y349. In some embodiments, the first Fc
polypeptide comprises the modifications T366W/S354C and the second
Fc polypeptide comprises the modifications T366S/L368A/Y407V/Y349C.
In some embodiments, the first Fc polypeptide comprises the
modifications L368D/K370S and the second Fc polypeptide comprises
the modifications S364K/E357Q.
[0033] In some embodiments, the first Fc polypeptide comprises the
modifications L368D/K370S and the second Fc polypeptide comprises
the modifications S364K/E357Q.
[0034] In some embodiments, at least one of the first and second
polypeptide comprises the modifications
Q295E/N384D/Q418E/N421D.
[0035] In some embodiments, one of the first or second Fc
polypeptide of the heterodimeric Fc further comprises a
modification at residue Ile253. In some instances, the modification
is Ile253Arg. In some embodiments, one of the first or second Fc
polypeptide of the heterodimeric Fc further comprises a
modification at residue His435. In some instances, the modification
is His435Arg.
[0036] In some embodiments, the Fc region, such as the first and/or
second Fc polypeptide comprises a polypeptide that lacks
Lys447.
[0037] In some of any of the provided embodiments, the first
polypeptide of the heterodimeric Fc comprises the sequence of amino
acids set forth in any of SEQ ID NOS:82, 86 or 201, and the second
polypeptide of the heterodimeric Fc comprises the sequence of amino
acids set forth in any of SEQ ID NOS:83, 87, 90, 92, 202 or 205. In
some embodiments, the first Fc polypeptide and the second Fc
polypeptide comprises sequences selected from the group consisting
of SEQ ID NOS: 82 and 83, respectively; SEQ ID NOS: 86 and 87,
respectively; SEQ ID NOS: 201 and 202, respectively; SEQ ID NOS: 82
and 90, respectively; SEQ ID NOS: 86 and 92, respectively; and SEQ
ID NOS: 201 and 205, respectively.
[0038] In some embodiments, the immunoglobulin Fc region is a
polypeptide comprising an amino acid sequence that is derived from
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 1-6 comprising one or modifications. In some embodiments, the
immunoglobulin Fc region is a polypeptide comprising an amino acid
sequence that is derived from an amino acid sequence selected from
the group consisting of SEQ ID NOs: 1-6 comprising one or
modifications to prevent glycosylation, to alter Fc receptor
interactions, to reduce Fc receptor binding, to enhance the
interaction with CD32A, to reduce the complement protein C1q
binding, to extend the half-life, to enhance FcRn binding, to alter
antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC), to induce
heterodimerization, to prevent dimerization, to stabilize the
homodimerization at the CH3:CH3 interface, and combinations
thereof.
[0039] In some embodiments, modifications within the Fc region
reduce binding to Fc-receptor-gamma receptors while having minimal
impact on binding to the neonatal Fc receptor (FcRn). In some
embodiments, the mutated or modified Fc polypeptide includes the
following mutations: Met252Tyr and Met428Leu or Met252Tyr and
Met428Val (M252Y, M428L, or M252Y, M428V) using the Kabat numbering
system.
[0040] In some embodiments, the Fc region comprises a polypeptide
comprising at least one modification to enhance FcRn binding. In
some examples, the modification is at a position selected from the
group consisting of Met252, Ser254, Thr256, Met428, Asn434, and
combinations thereof. In some cases, the modification is at a
position selected from the group consisting of Met252Y, Ser254T,
Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. In
some particular embodiments, the modification is at position Met252
and at position Met428. In some cases, the modification is Met252Y
and Met428L. In some cases, the modification is Met252Y and
Met428V.
[0041] In some embodiments, the first polypeptide of the
heterodimeric Fc comprises the sequence of amino acids set forth in
any of SEQ ID NOS: 94, 96 or 207, and the second polypeptide of the
heterodimeric Fc comprises the sequence of amino acids set forth in
any of SEQ ID NOS: 98, 100, or 209. In some embodiments, the first
Fc polypeptide and the second Fc polypeptide comprises sequences
selected from the group consisting of SEQ ID NOS: 94 and 98,
respectively; SEQ ID NOS: 96 and 100, respectively; and SEQ ID NOS:
207 and 209, respectively.
[0042] In some embodiments, the Fc region comprises a polypeptide
comprising at least one modification to enhance Fc.gamma.R binding.
In some cases, the modification is modification at Ser239 or
Ile332. In some embodiments, the glycosylation of the Fc region is
modified to enhance Fc.gamma.R binding as compared to an unmodified
Fc region. In some examples, the Fc region lacks or has reduced
fucose content.
[0043] In some embodiments, the Fc region comprises a polypeptide
comprising at least one amino acid modification that reduces
effector function and/or reduces binding to an effector molecule
selected from an Fc gamma receptor or C1q. In some embodiments, the
one or more amino acid modification is deletion of one or more of
Glu233, Leu234 or Leu235.
[0044] In some embodiments, the first polypeptide of the
heterodimeric Fc comprises the sequence of amino acids set forth in
any of SEQ ID NOS:82, 86, 94 or 96, and the second polypeptide of
the heterodimeric Fc comprises the sequence of amino acids set
forth in any of SEQ ID NOS:83, 87, 90, 92, 98 or 100. In some
embodiments, the Fc region comprises a polypeptide comprising at
least one amino acid modification that reduces effector function
and/or reduces binding to an effector molecule selected from an Fc
gamma receptor or C1q. In some examples, the one or more amino acid
modification is deletion of one or more of Glu233, Leu234 or
Leu235. In some aspects, the first polypeptide of the heterodimeric
Fc comprises the sequence of amino acids set forth in any of SEQ ID
NOS: 84, 88, 95 or 97 and the second polypeptide of the
heterodimeric Fc comprises the sequence of amino acids set forth in
any of SEQ ID NOS: 85, 89, 91, 93, 99 or 101.
[0045] In some embodiments, the first polypeptide of the
heterodimeric Fc comprises the sequence of amino acids set forth in
any of SEQ ID NOS: 84, 88, 95, 97, 203 or 208 and the second
polypeptide of the heterodimeric Fc comprises the sequence of amino
acids set forth in any of SEQ ID NOS: 85, 89, 91, 93, 99, 101, 204,
206 or 210. In some embodiments, the first Fc polypeptide and the
second Fc polypeptide comprises sequences selected from the group
consisting of SEQ ID NOS: 84 and 85, respectively; SEQ ID NOS: 88
and 89, respectively; SEQ ID NOS: 203 and 204, respectively; SEQ ID
NOS: 95 and 99, respectively; SEQ ID NOS: 97 and 101, respectively;
SEQ ID NOS: 208 and 210, respectively; SEQ ID NOS: 84 and 91,
respectively; SEQ ID NOS: 88 and 93, respectively; and SEQ ID NOS:
203 and 206, respectively.
[0046] In some embodiments, the CD3 binding region is an anti-CD3
antibody or antigen-binding fragment. In some embodiments, the
anti-CD3 antibody or antigen binding fragment comprises a variable
heavy chain region (VH) and a variable light chain region (VL). In
some of any such embodiments, the CD3 binding region is
monovalent.
[0047] In some embodiments, the anti-CD3 antibody or antigen
binding fragment is not a single chain antibody, optionally is not
a single chain variable fragment (scFv). In some embodiments, the
Fc is a heterodimeric Fc and the VH and VL that comprise the
anti-CD3 antibody or antigen binding fragment are linked to
opposite polypeptides of the heterodimeric Fc.
[0048] In some embodiments, the CD3 binding region is not able to,
or is not substantially able to, bind or engage CD3 unless at least
one of the antigen binding domains is bound to its TAA. In some
aspects, the CD3 binding region is not able to, or is not
substantially able, to bind or engage CD3 unless at least two of
the antigen binding domains is bound to their TAA(s).
[0049] In some embodiments, the multispecific polypeptide construct
contains a linker that is a polypeptide linker. In some
embodiments, the linker is a polypeptide of up to 25 amino acids in
length. In some cases, the linker is a polypeptide of from or from
about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino
acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino
acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids,
6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to
12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24
amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14
amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24
amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14
amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20
amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24
amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24
amino acids, 18 to 20 amino acids or 20 to 24 amino acids. In some
embodiments, the linker is a polypeptide that is 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in
length.
[0050] In some embodiments, the linker is 3 to 18 amino acids in
length. In some embodiments, the linker is 12 to 18 amino acids in
length. In some embodiments, the linker is 15 to 18 amino acids in
length. In some embodiments, the linker is a polypeptide that is 18
amino acids in length.
[0051] In some embodiments, the non-cleavable linker does not
contain a substrate recognition site that is specifically
recognized for cleavage by a protease. In some embodiments, the
protease is produced by an immune effector cell, by a tumor, or by
cells present in the tumor microenvironment. In some embodiments,
the protease is produced by an immune effector cell and the immune
effector cell is an activated T cell, a natural killer (NK) cell,
or an NK T cell. In some embodiments, the protease is selected from
among matriptase, a matrix metalloprotease (MMP), granzyme B, and
combinations thereof. In some embodiments, the protease is granzyme
B.
[0052] In some embodiments, the linker comprises the amino acid
sequence GS, GGS, GGGGS (SEQ ID NO:149), GGGGGS (SEQ ID NO:135) and
combinations thereof. In some embodiments, the linker comprises the
amino acid sequence (GGS)n, wherein n is 1 to 10. In some
embodiments, the linker comprises the amino acid sequence (GGGGS)n
(SEQ ID NO: 173), wherein n is 1 to 10. In some embodiments, the
linker comprises (GGGGGS)n (SEQ ID NO:172), wherein n is 1 to
4.
[0053] In some embodiments, the linker is or comprises GGS. In some
embodiments, the linker is or comprises GGGGS (SEQ ID NO: 149). In
some embodiments, the linker is or comprises GGGGGS (SEQ ID NO:
135). In some embodiments, the linker is or comprises GGSGGS
("(GGS).sub.2") (SEQ ID NO: 10). In some embodiments, the linker is
or comprises GGSGGSGGS ("(GGS).sub.3") (SEQ ID NO: 11). In some
embodiments, the linker is or comprises GGSGGSGGSGGS
("(GGS).sub.4") (SEQ ID NO: 12). In some embodiments, the linker is
or comprises GGSGGSGGSGGSGGS ("(GGS).sub.5") (SEQ ID NO: 13). In
some embodiments, the linker is or comprises GGGGGSGGGGGSGGGGGS
(SEQ ID NO: 119). In some embodiments, the linker is or comprises
GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147). In some embodiments, the
linker is or comprises GGGGSGGGGSGGGGS (SEQ ID NO:170). In some
embodiments, the linker is or comprises GGGGG (SEQ ID NO:192).
[0054] In some embodiments, the antigen binding domain and the
immunoglobulin Fc region of the first component (which in some
cases is the first antigen binding domain) are operably linked via
one or more further amino acid linkers (referred to herein as an
intra-component linker). The intra-component peptide linker of the
first component (also called LP1) can be a peptide linker such as
any as described in Section 11.3. The intra-component linker
present in the first component, i.e. linking the Fc region and an
antigen binding domain, can be of various lengths, for example 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids
in length. In some embodiments, these intra-component linkers are
composed predominately of the amino acids Glycine and Serine,
denoted as GS-linkers herein. In some embodiments, the GS-linker
comprises an amino acid sequence selected from the group consisting
of GGSGGS, i.e., (GGS).sub.2 (SEQ ID NO: 10); GGSGGSGGS, i.e.,
(GGS).sub.3 (SEQ ID NO: 11); GGSGGSGGSGGS, i.e., (GGS).sub.4 (SEQ
ID NO: 12); and GGSGGSGGSGGSGGS, i.e., (GGS).sub.5 (SEQ ID NO:
13).
[0055] In some embodiments, the multispecific polypeptide construct
comprises at least (i) a first polypeptide comprising the first Fc
polypeptide of the heterodimeric Fc region, the linker and the VH
domain of the anti-CD3 antibody or antigen binding fragment
thereof; and (ii) a second polypeptide comprising the second Fc
polypeptide of the heterodimeric Fc region, the linker and the VL
domain of the anti-CD3 antibody or antigen binding fragment
thereof, wherein one or both of the first and second polypeptide
comprise at least one antigen-binding domain that binds to a tumor
associated antigen (TAA). In some embodiments, the VH of the
anti-CD3 antibody or antigen-binding fragment is on the same
polypeptide as the at least one antigen-binding domain that binds
to a tumor associated antigen (TAA). In some embodiments, the
polypeptide comprising the VL of the anti-CD3 antibody or
antigen-binding fragment does not contain the at least one
antigen-binding domain that binds to a tumor associated antigen
(TAA). In particular embodiments of provided multispecific
polypeptide constructs, at least one antigen binding domain is
positioned carboxy-terminally relative to the CD3 binding region of
the multispecific polypeptide construct.
[0056] In some embodiments, the second component includes one or
more copies of the CD3 binding region.
[0057] In some embodiments, the CD3 binding region is an anti-CD3
antibody or antigen-binding fragment that includes one or more
copies of an antibody or an antigen-binding fragment thereof that
is able to bind or engage CD3, such as CD3.epsilon.. In some
embodiments, the anti-CD3 binding domain includes one or more
copies of an antibody or an antigen-binding fragment thereof
selected from the group consisting of a Fab fragment, a
F(ab').sub.2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a
single domain heavy chain antibody, and a single domain light chain
antibody.
[0058] In some embodiments, the anti-CD3 binding domain includes an
Fv antibody fragment that binds CD3.epsilon. (referred to herein as
an anti-CD3.epsilon. Fv fragment).
[0059] In some embodiments, the anti-CD3.epsilon. Fv antibody
fragment includes an amino acid sequence selected from the group of
SEQ ID NO: 32-81, 191, 196-200, 211, and 212. In some embodiments,
the anti-CD3.epsilon. Fv antibody fragment includes an amino acid
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to an amino acid sequence selected from
the group consisting of SEQ ID NO: 32-81, 191, 196-200, 211, and
212. In some embodiments, the anti-CD3.epsilon. Fv antibody
fragment includes a combination of an amino acid sequence selected
from the group of SEQ ID NO: 32-62, 196-198, and 211 and an amino
acid sequence selected from the group consisting of SEQ ID NO:
63-81, 191, 199, 200, and 212. In some embodiments, the
anti-CD3.epsilon. Fv antibody fragment includes a combination of an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to an amino acid sequence
selected from the group consisting of SEQ ID NO: 32-62, 196-198,
and 211 and an amino acid sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino
acid sequence selected from the group consisting of SEQ ID NO:
63-81, 191, 199, 200, and 212.
[0060] In some embodiments, the anti-CD3.epsilon. Fv antibody
fragment is a disulfide stabilized anti-CD3 binding Fv fragment
(dsFv).
[0061] In some embodiments, the first component includes one or
more copies of an antigen-binding domain. In certain embodiments,
the first component contains at least two antigen binding domains,
such as two antigen binding domains. In some embodiments, the at
least two antigen binding domains of the first component bind to
the same TAA. In some cases, the at least two antigen binding
domains of the first component bind to different epitopes of the
same TAA. In some instances, the at least two antigen binding
domains of the first component bind to the same epitope of the same
TAA. In some embodiments, the at least two antigen binding domain
of the first component bind to different TAAs.
[0062] In some embodiments, the second component includes one or
more copies of an antigen-binding domain. In certain embodiments,
the second component contains at least two antigen binding domains,
such as two antigen binding domains. In some embodiments, the at
least two antigen binding domains of the second component bind to
the same TAA. In some cases, the at least two antigen binding
domains of the second component bind to different epitopes of the
same TAA. In some instances, the at least two antigen binding
domains of the second component binds to a same epitope of the same
TAA. In some embodiments, the at least two antigen binding domains
of the second component bind to different TAAs.
[0063] In some embodiments, the first component contains a first
antigen binding domain and the antigen binding domain of the second
component is a second antigen binding domain. In some embodiments,
the multispecific antigen binding domain comprises at least a first
antigen binding domain and a second antigen binding domain, wherein
the first antigen binding domain and second antigen binding domain
bind to the same TAA. In some cases, the first antigen binding
domain and the second antigen binding domain bind different
epitopes of the same TAA. In some instances, the first antigen
binding domain and the second antigen binding domain bind the same
epitope of the same TAA. In some embodiments, the multispecific
antigen binding domain comprises at least a first antigen binding
domain and a second antigen binding domain wherein the first
antigen binding domain and the second antigen binding domain bind
different TAAs.
[0064] In some embodiments, the antigen binding domain of the
second component (which in some cases is the second antigen binding
domain) and the CD3 binding region are operably linked via one or
more further amino acid linkers (referred to herein as an
intra-component linker). The intra-component peptide linker of the
second component (also called LP2) can be a peptide linker such as
any as described in Section 11.3. The intra-component linker of
present in the second component, i.e. linking the CD3 binding
region and an antigen binding domain, can be of various lengths,
for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20 amino acids in length. In some embodiments, the intra-component
linker of the second component is composed predominately of the
amino acids Glycine and Serine, denoted as GS-linkers herein. In
some embodiments, the GS-linker comprises an amino acid sequence
selected from the group consisting of GGSGGS, i.e., (GGS).sub.2
(SEQ ID NO: 10); GGSGGSGGS, i.e., (GGS).sub.3 (SEQ ID NO: 11);
GGSGGSGGSGGS, i.e., (GGS).sub.4 (SEQ ID NO: 12); and
GGSGGSGGSGGSGGS, i.e., (GGS).sub.5 (SEQ ID NO: 13).
[0065] Provided herein is a multispecific polypeptide construct,
the multispecific polypeptide construct comprising a first
component comprising a heterodimeric Fc region and a second
component comprising an anti-CD3 antibody or antigen-binding
fragment comprising a variable heavy chain region (VH) and a
variable light chain region (VL), wherein: the VH and VL that
comprise the anti-CD3 antibody or antigen binding fragment are
linked to opposite polypeptides of the heterodimeric Fc; the first
and second components are coupled by a linker, wherein the
heterodimeric Fc region is positioned N-terminal to the anti-CD3
antibody or antigen-binding fragment; and one or both of the first
and second components comprises an antigen binding domain that
binds a tumor associated antigen (TAA).
[0066] In some embodiments, the linker is a polypeptide of up to 50
amino acids in length. In some embodiments, the linker is a
polypeptide of up to 25 amino acids in length. In some embodiments,
the linker is a polypeptide of up to 15 amino acids in length.
[0067] In any of the provide embodiments, the one or more antigen
binding domain that binds TAA results in monovalent, bivalent,
trivalent, or tetravalent binding to the TAA. In some embodiments,
the one or more antigen binding domains that bind TAA independently
are selected from an sdAb, an scFv or a Fab. In some embodiments,
the one or more antigen binding domains that binds a TAA is a
single chain molecule, such as a single chain antibody fragment
containing a VH and a VL, for example an sdAb or an scFv. In some
embodiments the one or more antigen binding domains that binds a
TAA is a sdAb, such as a V.sub.HH or a VH.sub.NAR. In some
embodiments, at least one of the antigen binding domains is a Fab
containing a first chain comprising a VH-CH1 (Fd) and a second
chain comprising a VL-CL.
[0068] In some embodiments, the antigen binding domain that binds
the TAA is attached to the VH of the anti-CD3 binding domain. In
some embodiments, the antigen binding domain that binds the TAA is
attached to the same side (e.g., knob or hole) of the heterodimeric
Fc to which the VH of the anti-CD3 binding domain is attached. In
some embodiments, the antigen binding domain that binds the TAA is
a sdAb attached to the VH of the anti-CD3 binding domain. In some
embodiments, the antigen binding domain that binds the TAA is a
sdAb attached to same side (e.g., knob or hole) of the
heterodimeric Fc domain to which the VH of the anti-CD3 binding
domain is attached. In some embodiments, the antigen binding domain
that binds the TAA is a V.sub.HH or a VH.sub.NAR that is attached
to the VH of the anti-CD3 binding domain. In some embodiments, the
antigen binding domain that binds the TAA is a V.sub.HH or a
VH.sub.NAR that is attached to the same side (e.g., knob or hole)
of the heterodimeric Fc domain to which the VH of the anti-CD3
binding domain is attached. In some embodiments, the antigen
binding domain that binds the TAA is a V.sub.HH attached to the VH
of the anti-CD3 binding domain. In some embodiments, the antigen
binding domain that binds the TAA is a V.sub.HH attached to the
same side (e.g., knob or hole) of the heterodimeric Fc domain to
which the VH of the anti-CD3 binding domain is attached. In some
embodiments, the antigen binding domain that binds the TAA is a
VH.sub.NAR attached to the VH of the anti-CD3 binding domain. In
some embodiments, the antigen binding domain that binds the TAA is
a VH.sub.NAR attached to the same side (e.g., knob or hole) or the
Fc domain to which the VH of the anti-CD3 binding domain is
attached.
[0069] In some embodiments, the multispecific polypeptide construct
comprises at least (i) a first polypeptide comprising the first Fc
polypeptide of the heterodimeric Fc region, the linker and the VH
domain of the anti-CD3 antibody or antigen binding fragment; and
(ii) a second polypeptide comprising the second Fc polypeptide of
the heterodimeric Fc region, the linker and the VL domain of the
anti-CD3 antibody or antigen binding fragment, wherein one or both
of the first and second polypeptide comprise at least one
antigen-binding domain that binds to a tumor associated antigen
(TAA). In some instances, only one of the first or second
polypeptide comprises the at least one antigen-binding domain that
binds a TAA.
[0070] In some embodiments, the at least one of the antigen binding
domain(s) is a Fab. In some embodiments, the multispecific
polypeptide construct comprises: (i) a first polypeptide comprising
the first Fc polypeptide of the heterodimeric Fc region, the linker
and the VH domain of the anti-CD3 antibody or antigen binding
fragment; (ii) a second polypeptide comprising the second Fc
polypeptide of the heterodimeric Fc region, the linker and the VL
domain of the anti-CD3 antibody or antigen binding fragment, and
(iii) a third polypeptide comprising a VH-CH1 (Fd) or VL-CL of a
Fab antibody fragment that binds to a tumor-associated antigen,
wherein the first and/or second polypeptide further comprises the
other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. In
some cases, only one of the first or second polypeptide comprises
the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment.
In some embodiments, both the first or second polypeptide comprises
the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment.
In some cases, the other of the VH-CH1 (Fd) or VL-CL of the Fab
antibody fragment is positioned amino-terminally relative to the Fc
region and/or carboxy-terminally relative to the CD3 binding region
of one of the first or second polypeptide of the multispecific
polypeptide construct. In some embodiments, the other of the VH-CH1
(Fd) or VL-CL of the Fab antibody fragment is positioned
amino-terminally relative to the Fc region of the first polypeptide
or second polypeptide and carboxy-terminally relative to the CD3
binding region of the other of the first or second polypeptide.
[0071] In some embodiments, the at least one antigen binding domain
is positioned amino-terminally relative to the Fc region and/or is
positioned carboxy-terminally relative to the CD3 binding region of
one of the first or second polypeptide of the multispecific
polypeptide construct. In some cases, the at least one antigen
binding domain is positioned amino-terminally relative to the Fc
region of the multispecific construct and the second antigen
binding domain is positioned carboxy-terminally relative to the CD3
binding region of the multispecific construct. In particular
embodiments of provided multispecific polypeptide constructs, at
least one antigen binding domain is positioned carboxy-terminally
relative to the CD3 binding region of the multispecific polypeptide
construct. In some embodiments, the at least one antigen binding
domain is a sdAb. In some embodiments the at least one antigen
binding domain that is a sdAb is positioned carboxy-terminally to
the CD3 binding region of the multispecific construct. In some
embodiments the at least one antigen binding domain that is a sdAb
is positioned amino-terminally to the Fc region of the
multispecific construct. In some embodiments the at least one
antigen binding domain is a V.sub.HH. In some embodiments, the at
least one antigen binding domain that is a V.sub.HH is positioned
carboxy-terminally to the CD3 binding region of the multispecific
construct. In some embodiments the at least one antigen binding
domain that is a V.sub.HH is positioned amino-terminally to the Fc
region of the multispecific construct.
[0072] In some embodiments, the multispecific polypeptide construct
comprises a first linking peptide (LP1) between the first antigen
binding domain and the immunoglobulin Fc polypeptide region (Fc
region). In some embodiments, the multispecific polypeptide
construct comprises a second linking peptide (LP2) between the
anti-CD3 binding domain (CD3 binding region) and the second antigen
binding domain. In some embodiments, the multispecific polypeptide
construct comprises a first linking peptide (LP1) between the first
antigen binding domain and the immunoglobulin Fc polypeptide region
(Fc region) and a second linking peptide (LP2) between the anti-CD3
binding domain (CD3 binding region) and the second antigen binding
domain.
[0073] In some embodiments, the multispecific polypeptide construct
has the structural arrangement from N-terminus to C-terminus as
follows: first antigen binding domain-LP1-immunoglobulin Fc
polypeptide linker region (Fc region)-linker-anti-CD3 binding
domain-LP2-second antigen binding domain. In some embodiments, the
multispecific polypeptide construct has the structural arrangement
from N-terminus to C-terminus as follows: second antigen binding
domain-LP2-immunoglobulin Fc polypeptide linker region (Fc
region)-linker-anti-CD3 binding domain (CD3 binding
region)-LP1-first antigen binding domain.
[0074] In some embodiments, the two linking peptides LP1 and LP2
are not identical to each other. In some cases, LP1 or LP2 is
independently a peptide of about 1 to 20 amino acids in length. In
some examples, LP1 or LP2 independently comprise a peptide that is
or comprises any Gly-Ser linker as set forth in SEQ ID NOs: 10-13,
119, 135, 147, 149.
[0075] In some embodiments, the multispecific construct is a
construct having any of the structural arrangement shown in FIG. 1.
In some embodiments, the construct is a bispecific construct that
has a structural arrangement from N-terminus to C-terminus as
follows. The N-terminal end of the bispecific construct includes a
first antigen binding domain that binds a tumor associated antigen
(TAA). The first binding domain binds a first epitope on the TAA
target. Coupled to the first antigen binding domain is a central
immunoglobulin Fc polypeptide region that regulates Fc.gamma.R
interactions and/or FcRn interaction. In some embodiments, the
central immunoglobulin Fc polypeptide region is heterodimeric. The
immunoglobulin Fc polypeptide region is coupled to a linker located
at a position C-terminal to the end of the immunoglobulin Fc
polypeptide region. The linker is attached to an anti-CD3 binding
sequence located C-terminal to the Fc region, which, in some cases,
is at the distal end of the second component. The C-terminus of the
bispecific construct includes a second antigen binding domain that
binds a TAA. In some embodiments, the second antigen binding domain
binds the same TAA as the first antigen binding domain located
within the first component. In some embodiments, the second antigen
binding domain binds a second epitope on the TAA, wherein the
second epitope is non-competitive with the first epitope on the
TAA. In some embodiments, the second antigen binding domain binds a
distinct TAA from that of the first antigen binding domain.
[0076] In some of any of the provided embodiments, the anti-CD3
antibody or antigen binding fragment is an Fv antibody fragment. In
some embodiments, the Fv antibody fragment comprises a disulfide
stabilized anti-CD3 binding Fv fragment (dsFv). In some
embodiments, the anti-CD3 binding sequence is an Fv antibody
fragment that is engineered to include a disulfide linkage between
the variable heavy chain (VH) and variable light chain (VL)
regions, thereby producing a disulfide stabilized anti-CD3 binding
Fv fragment (dsFv). In some embodiments, the VH and VL domains that
comprise the anti-CD3 Fv are operably linked to opposite members of
a heterodimeric Fc region. In these embodiments, the anti-CD3 Fv
binds CD3 in a monovalent fashion. In aspects as provided, the
anti-CD3 dsFv does not engage CD3 unless the multispecific
polypeptide construct is bound to a cognate antigen.
[0077] In some embodiments, each of the first antigen binding
domain and the second antigen binding domain of the bispecific
construct includes one or more copies of an antibody or an
antigen-binding fragment thereof. In some embodiments, each of the
first antigen binding domain and the second antigen binding domain
of the bispecific construct includes one or more copies of an
antibody or an antigen-binding fragment thereof selected from the
group consisting of a Fab fragment, a F(ab').sub.2 fragment, an Fv
fragment, a scFv, a scAb, a dAb, a single domain heavy chain
antibody, and a single domain light chain antibody. In some
embodiments, the antigen binding domain, or independently each of
the antigen binding domains, is an antibody or antigen-binding
fragment thereof selected from the group consisting of a Fab
fragment, a F(ab')2 fragment, an Fv fragment, a scFv, a scAb, a
dAb, a single domain heavy chain antibody, and a single domain
light chain antibody. In some embodiments, the each of the first
antigen binding domain and the second antigen binding domain of the
bispecific construct includes one or more copies of one or more
single domain antibody (sdAb) fragments, for example V.sub.HH,
V.sub.NAR, engineered V.sub.H or V.sub.K domains. V.sub.HHs can be
generated from natural camelid heavy chain only antibodies,
genetically modified rodents that produce heavy chain only
antibodies, or naive/synthetic camelid or humanized camelid single
domain antibody libraries. V.sub.NAR5 can be generated from
cartilaginous fish heavy chain only antibodies. Various methods
have been implemented to generate monomeric sdAbs from
conventionally heterodimeric V.sub.H and V.sub.K domains, including
interface engineering and selection of specific germline
families.
[0078] In some embodiments, the antibody or antigen-binding
fragment is an sdAb. In some cases, the sdAb is a human or
humanized sdAb. In some aspects, the sdAb is V.sub.HH, V.sub.NAR,
an engineered VH domain or an engineered VK domain. In some
examples, the antibody or antigen-binding fragment thereof is an
scFv. In some cases, the antibody or antigen-binding fragment
thereof is a Fab.
[0079] In some of any of the provided embodiments, the anti-CD3
antibody or antigen-binding fragment comprises a VH CDR1 comprising
the amino acid sequence TYAMN (SEQ ID NO: 16); a VH CD2 comprising
the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH
CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:
18), a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN
(SEQ ID NO: 19); a VL CDR2 comprising the amino acid sequence
GTNKRAP (SEQ ID NO: 20); and a VL CDR3 comprising the amino acid
sequence ALWYSNLWV (SEQ ID NO: 21).
[0080] In some of any of the provided embodiments, the anti-CD3
antibody or antigen-binding fragment comprises a VH CDR1 comprising
the amino acid sequence GFTFNTYAMN (SEQ ID NO: 211); a VH CDR2
comprising the amino acid sequence RIRSKYNNYATY (SEQ ID NO:212); a
VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID
NO: 18), a VL CDR1 comprising the amino acid sequence
RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 comprising the amino acid
sequence GTNKRAP (SEQ ID NO: 20); and a VL CDR3 comprising the
amino acid sequence ALWYSNLWV (SEQ ID NO: 21).
[0081] In some of any of the provided embodiments, the anti-CD3
antibody or antigen-binding fragment comprises a VH CDR1 sequence
that includes at least the amino acid sequence GFTFNTYAMN (SEQ ID
NO: 211); a VH CDR2 sequence that includes at least the amino acid
sequence RIRSKYNNYATY (SEQ ID NO: 212); a VH CDR3 sequence that
includes at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID
NO: 18), a VL CDR1 sequence that includes at least the amino acid
sequence GSSTGAVTTSNYAN (SEQ ID NO: 229); a VL CDR2 sequence that
includes at least the amino acid sequence GTNKRAP (SEQ ID NO: 230);
and a VL CDR3 sequence that includes at least the amino acid
sequence ALWYSNHWV (SEQ ID NO: 225).
[0082] In some of any of the provided embodiments, the anti-CD3
antibody or antigen-binding fragment comprises: a VH having the
amino acid sequence of any of SEQ ID NOS: 14, 32-62, 196-198, and
211 or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS:
14, 32-62, 196-198, and 211; and a VL having the amino acid
sequence of any of SEQ ID NOS: 15, 63-81, 191, 199, 200, and 212 or
a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS: 15,
63-81, 191, 199, 200, and 212.
[0083] In some embodiments, the anti-CD3 antibody or
antigen-binding fragment is an Fv. In some embodiments, the
anti-CD3 Fv comprises: a VH having the amino acid sequence of any
of SEQ ID NOS: 14, 32-43, 45-47, 48, 196 and 211 or a sequence that
exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to any of SEQ ID NOS: 14, 32-43, 45-47, 48,
196 and 211; and a VL having the amino acid sequence of any of SEQ
ID NOS: 15, 63, 65-71, 73, 75, 77, and 199 or a sequence that
exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to any of SEQ ID NOS: 15, 63, 65-71, 73, 75,
77, and 199. In some cases, the anti-CD3 dsFv comprises the amino
acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ
ID NO: 15. In other cases, the anti-CD3 Fv comprises the amino acid
sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID
NO:199.
[0084] In some embodiments, the VH and VL chain regions of the CD3
binding domain each independently comprise at least one amino acid
modification. In some embodiments, the at least one amino acid
modification of the VH and VL chain regions of the CD3 binding
domain increase the stability of the CD3 binding domain. In some
embodiments, the at least one amino acid modification of the VH and
VL chain regions of the CD3 binding domain increase the ability of
the CD3 binding domain to bind CD3. In some embodiments, the at
least one amino acid modification of the VH and VL chain regions of
the CD3 binding domain increase the stability of the CD3 binding
domain by creating a disulfide linkage between the VH and VL chain
regions.
[0085] In some embodiments, the CD3 binding region has a disulfide
stabilized linkage between the VH and VL regions. In some
embodiments, the anti-CD3 antibody or antigen-binding fragment is
disulfide stabilized Fv (dsFv). In some embodiments, the disulfide
stabilized anti-CD3 Fv comprises an anti-CD3 VH with the mutation
44 to Cys and an anti-CD3 VL with the mutation 100 to Cys by Kabat
numbering. In some embodiments, the disulfide stabilized anti-CD3
Fv comprises an anti-CD3 VH with the mutation G44C and an anti-CD3
VL with the mutation G100C by Kabat numbering. In some embodiments,
the disulfide stabilized anti-CD3 Fv comprises an anti-CD3 VH with
the mutation at position 105 to Cys and an anti-CD3 VL with the
mutation position 43 to Cys by Kabat numbering.
[0086] In some embodiments, the anti-CD3 dsFv comprises: a VH
having the amino acid sequence of any of SEQ ID NOS: 44, 49-62, 197
and 198 or a sequence that exhibits at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID
NOS: 44, 49-62, 197 and 198; and a VL having the amino acid
sequence of any of SEQ ID NOS: 64, 72, 74, 76, 78-81, 191, 200 and
212 or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS:
64, 72, 74, 76, 78-81, 191, 200 and 212. In some cases, the
anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 44
and the amino acid sequence of SEQ ID NO: 72. In some embodiments,
the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:
198 and the amino acid sequence of SEQ ID NO: 200. In some
embodiments, the anti-CD3 dsFv comprises the amino acid sequence of
SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 200.
[0087] In some embodiments, the multispecific construct also
includes an agent conjugated to the multispecific construct. In
some embodiments, the agent is a therapeutic agent. In some
embodiments, the agent is a detectable moiety. In some embodiments,
the detectable moiety is a diagnostic agent. In some embodiments,
the agent is conjugated to the multispecific construct via a
linker. In some embodiments, the linker is a non-cleavable
linker.
[0088] In some embodiments, the multispecific construct described
herein is used in conjunction with one or more additional agents or
a combination of additional agents. Suitable additional agents
include current pharmaceutical and/or surgical therapies for an
intended application, such as, for example, cancer. For example,
the multispecific construct can be used in conjunction with an
additional chemotherapeutic or anti-neoplastic agent.
[0089] In some embodiments, the multispecific construct and
additional agent are formulated into a single therapeutic
composition, and the multispecific construct and additional agent
are administered simultaneously. In some embodiments, the
multispecific construct and additional agent are separate from each
other, e.g., each is formulated into a separate therapeutic
composition, and the multispecific construct and the additional
agent are administered simultaneously, or the multispecific
construct and the additional agent are administered at different
times during a treatment regimen. For example, the multispecific
construct is administered prior to the administration of the
additional agent, the multispecific construct is administered
subsequent to the administration of the additional agent, or the
multispecific construct and the additional agent are administered
in an alternating fashion. As described herein, the multispecific
construct and additional agent are administered in single doses or
in multiple doses.
[0090] In some embodiments, the multispecific construct naturally
contains one or more disulfide bonds. In some embodiments, the
multispecific construct can be engineered to include one or more
disulfide bonds.
[0091] The disclosure also provides an isolated nucleic acid
molecule or polynucleotide encoding at least a portion of a
multispecific construct described herein and/or one or more nucleic
acid molecules encoding a multispecific construct described herein,
such as for example, at least a first nucleic acid encoding at
least a portion of the first component of the multispecific
construct and a second nucleic acid encoding at least a portion of
the second component of the multispecific construct, as well as
vectors that include these isolated nucleic acid sequences.
[0092] Among the provided embodiments is a polynucleotide(s)
encoding any of the provided multispecific polypeptide constructs.
Also provided is a polynucleotide encoding a polypeptide chain of
any of the provided multispecific polypeptide constructs. Further
provided is a polynucleotide, comprising a first nucleic acid
sequence encoding a first polypeptide of any of the provided
multispecific constructs and a second nucleic acid sequence
encoding a second polypeptide of the multispecific construct,
wherein the first and second nucleic acid sequence are separated by
an internal ribosome entry site (IRES), or a nucleic acid encoding
a self-cleaving peptide or a peptide that causes ribosome skipping.
In some cases, the first nucleic acid sequence and second nucleic
acid sequence are operably linked to the same promoter. In some
embodiments, the multispecific polypeptide construct comprises a
third polypeptide chain, and the polynucleotide further comprises a
third nucleic acid encoding the third polypeptide of the
multispecific construct. In some embodiments, the third nucleic
acid is separated from the first and/or second polypeptide by an
internal ribosome entry site (IRES), or a nucleic acid encoding a
self-cleaving peptide or a peptide that causes ribosome skipping
and/or the third nucleic acid sequence is operably linked to the
same promoter as the first and/or second nucleic acid sequence. In
some examples, the nucleic acid encoding a self-cleaving peptide or
a peptide that causes ribosome skipping is selected from a T2A, a
P2A, a E2A or a F2A (SEQ ID NOS: 159-164, or encoded by the
sequence set forth in SEQ ID NO: 165)
[0093] Provided herein is a vector comprising any of the provided
polynucleotides. In some embodiments, the vector is an expression
vector. In some examples, the vector is a viral vector or a
eukaryotic vector, optionally wherein the eukaryotic vector is a
mammalian vector.
[0094] Provided is a cell, comprising any of the provided
polynucleotides or vectors. In some cases, the cell is recombinant
or isolated. In some examples, the cell is a mammalian cell. In
some examples, the cell is a HEK293 or CHO cell.
[0095] The disclosure provides methods of producing a multispecific
construct by culturing a cell under conditions that lead to
expression of the multispecific construct, wherein the cell
comprises such a nucleic acid molecule(s). In some embodiments, the
cell comprises such a vector.
[0096] Provided herein is a method of producing a multispecific
polypeptide construct, the method comprising introducing into a
cell any of the provided polynucleotides or vectors and culturing
the cell under conditions to that lead to expression of the
multispecific construct to produce the multispecific polypeptide
construct. Also provided is a method of producing a multispecific
polypeptide construct, the method comprising culturing any of the
provided cells under conditions in which the multispecific
polypeptide is expressed or produced by the cell. In some cases,
the cell is a mammalian cell. In some examples, the cell is a
HEK293 or CHO cell. In some embodiments, the method further
includes isolating or purifying the multispecific polypeptide
construct from the cell. In some cases, the multispecific
polypeptide construct is a heterodimer.
[0097] Provided herein is a multispecific polypeptide construct
produced by any of the provided methods.
[0098] Provided herein is a method of stimulating or inducing an
immune response, the method comprising contacting a target cell and
a T cell with the any of the provided multispecific polypeptide
constructs or pharmaceutical compositions, said target cell
expressing a tumor associated antigen recognized by the
multispecific polypeptide construct. In some embodiments, the
target cell is a tumor cell expressing the tumor associated antigen
(TAA).
[0099] In some embodiments, the contacting is carried out ex vivo
or in vitro. In some embodiments, the contacting is carried out in
vivo in a subject.
[0100] Provided is a method of stimulating or inducing an immune
response in a subject, the method comprising administering, to a
subject in need thereof, a therapeutically effective amount of any
of the provided multispecific conjugates or pharmaceutical
compositions. In some cases, the method increases cell-mediated
immunity. In some embodiments, the method increases T-cell
activity. In some embodiments, the method increases cytolytic
T-cell (CTL) activity. In some examples, the immune response is
increased against a tumor or cancer. In some embodiments, the
method treats a disease or condition in the subject.
[0101] The present disclosure also provides methods of treating,
preventing, delaying the progression of or otherwise ameliorating a
symptom of one or more pathologies or alleviating a symptom
associated with such pathologies, by administering a multispecific
polypeptide construct of the disclosure to a subject in which such
treatment or prevention is desired. Provided herein is a method of
treating a disease or condition in a subject, the method comprising
administering, to a subject in need thereof, a therapeutically
effective amount of any of the provided multispecific conjugates or
pharmaceutical compositions. In some embodiments, the disease or
condition is a tumor or a cancer.
[0102] In some embodiments of any of the provided method, the
subject, such as the subject to be treated is, e.g., human or other
mammal. In some embodiments of any of the provided method, the
subject is a human. In some embodiments, the subject is a non-human
mammal, such as a non-human primate, companion animal (e.g., cat,
dog, horse), farm animal, work animal, or zoo animal. In some
embodiments, the subject is a rodent.
[0103] A multispecific polypeptide construct of the disclosure used
in any of the embodiments of these methods and uses can be
administered at any stage of the disease. For example, such a
multispecific polypeptide construct can be administered to a
patient suffering cancer of any stage, from early to metastatic.
The terms subject and patient are used interchangeably herein.
[0104] A multispecific polypeptide construct of the disclosure used
in any of the embodiments of these methods and uses can be used in
a treatment regimen comprising neoadjuvant therapy.
[0105] A multispecific polypeptide construct of the disclosure used
in any of the embodiments of these methods and uses can be
administered either alone or in combination with one or more
additional agents, including small molecule inhibitors, other
antibody-based therapies, polypeptide or peptide-based therapies,
nucleic acid-based therapies and/or other biologics. In some
embodiments, a multispecific polypeptide construct is administered
in combination with one or more additional agents such as, by way
of non-limiting example, a chemotherapeutic agent, such as an
alkylating agent, an anti-metabolite, an anti-microtubule agent, a
topoisomerase inhibitor, a cytotoxic antibiotic, and any other
nucleic acid damaging agent. In some embodiments, the additional
agent is a taxane, such as paclitaxel (e.g., Abraxane.RTM.). In
some embodiments, the additional agent is an anti-metabolite, such
as gemcitabine. In some embodiments, the additional agent is an
alkylating agent, such as platinum-based chemotherapy, such as
carboplatin or cisplatin. In some embodiments, the additional agent
is a targeted agent, such as a kinase inhibitor, e.g., sorafenib or
erlotinib. In some embodiments, the additional agent is a targeted
agent, such as another antibody, e.g., a monoclonal antibody (e.g.,
bevacizumab), a bispecific antibody, or a multispecific antibody.
In some embodiments, the additional agent is a proteasome
inhibitor, such as bortezomib or carfilzomib. In some embodiments,
the additional agent is an immune modulating agent, such as
lenolidominde or IL-2. In some embodiments, the additional agent is
radiation. In some embodiments, the additional agent is an agent
considered standard of care by those skilled in the art. In some
embodiments, the additional agent is a chemotherapeutic agent well
known to those skilled in the art. In some embodiments, the
multispecific polypeptide construct and the additional agent(s) are
formulated in a single composition. In some embodiments, the
multispecific polypeptide construct and the additional agent(s) are
administered as two or more separate compositions. In some
embodiments, the multispecific polypeptide construct and the
additional agent(s) are administered simultaneously. In some
embodiments, the multispecific polypeptide construct and the
additional agent(s) are administered sequentially.
[0106] In some embodiments, the additional agent(s) is a
chemotherapeutic agent, such as a chemotherapeutic agent selected
from the group consisting of docetaxel, paclitaxel, abraxane (i.e.,
albumin-conjugated paclitaxel), doxorubicin, oxaliplatin,
carboplatin, cisplatin, irinotecan, and gemcitabine.
[0107] In some embodiments, the additional agent(s) is a checkpoint
inhibitor, a kinase inhibitor, an agent targeting inhibitors in the
tumor microenvironment, and/or a T cell or NK agonist. In some
embodiments, the additional agent(s) is radiation therapy, alone or
in combination with another additional agent(s) such as a
chemotherapeutic or anti-neoplastic agent. In some embodiments, the
additional agent(s) is a vaccine, an oncovirus, and/or a
DC-activating agent such as, by way of non-limiting example, a
toll-like receptor (TLR) agonist and/or .alpha.-CD40. In some
embodiments, the additional agent(s) is a tumor-targeted antibody
designed to kill the tumor via ADCC or via direct conjugation to a
toxin (e.g., an antibody drug conjugate (ADC).
[0108] In some embodiments, the checkpoint inhibitor is an
inhibitor of a target selected from the group consisting of CTLA-4,
LAG-3, PD-1, PDL1, TIGIT, TIM-3, B7H3, B7H4, and Vista. In some
embodiments, the kinase inhibitor is selected from the group
consisting of B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib.
In some embodiments, the kinase inhibitor is crizotinib. In some
embodiments, the tumor microenvironment inhibitor is selected from
the group consisting of an IDO inhibitor, an .alpha.-CSF1R
inhibitor, an .alpha.-CCR4 inhibitor, a TGF-beta, a myeloid-derived
suppressor cell, or a T-regulatory cell. In some embodiments, the
agonist is selected from the group consisting of OX40, GITR, CD137,
CD28, ICOS, CD27, and HVEM. In some embodiments, the checkpoint
inhibitor is an antibody that binds a target selected from CTLA-4,
PD-1, and/or PD-L1. In some embodiments, the checkpoint inhibitor
is an anti-CTLA4 antibody, an anti-PD-1 antibody, and an anti-PD-L1
antibody, and/or combinations thereof. In some embodiments, the
checkpoint inhibitor is an anti-CTLA4 antibody such as, e.g.,
Yervoy.TM. In some embodiments, the checkpoint inhibitor is an
anti-PD-1 antibody such as, e.g., Opdivo.TM. and/or
Keytruda.TM..
[0109] In some embodiments, the inhibitor is a CTLA-4 inhibitor. In
some embodiments, the inhibitor is a LAG-3 inhibitor. In some
embodiments, the inhibitor is a PD-1 inhibitor. In some
embodiments, the inhibitor is a PDL1 inhibitor. In some
embodiments, the inhibitor is a TIGIT inhibitor. In some
embodiments, the inhibitor is a TIM-3 inhibitor. In some
embodiments, the inhibitor is a B7H3 inhibitor. In some
embodiments, the inhibitor is a B7H4 inhibitor. In some
embodiments, the inhibitor is a Vista inhibitor. In some
embodiments, the inhibitor is a B-RAFi inhibitor. In some
embodiments, the inhibitor is a MEKi inhibitor. In some
embodiments, the inhibitor is a Btk inhibitor. In some embodiments,
the inhibitor is ibrutinib. In some embodiments, the inhibitor is
crizotinib. In some embodiments, the inhibitor is an IDO inhibitor.
In some embodiments, the inhibitor is an .alpha.-CSF1R inhibitor.
In some embodiments, the inhibitor is an .alpha.-CCR4 inhibitor. In
some embodiments, the inhibitor is a TGF-beta. In some embodiments,
the inhibitor is a myeloid-derived suppressor cell. In some
embodiments, the inhibitor is a T-regulatory cell.
[0110] In some embodiments, the agonist is OX40. In some
embodiments, the agonist is GITR. In some embodiments, the agonist
is CD137. In some embodiments, the agonist is CD28. In some
embodiments, the agonist is ICOS. In some embodiments, the agonist
is CD27. In some embodiments, the agonist is HVEM.
[0111] In some embodiments, the multispecific polypeptide construct
is administered during and/or after treatment in combination with
one or more additional agents such as, for example, a
chemotherapeutic agent, an anti-inflammatory agent, and/or a an
immunosuppressive agent. In some embodiments, the multispecific
polypeptide construct and the additional agent are formulated into
a single therapeutic composition, and the multispecific polypeptide
construct and additional agent are administered simultaneously.
Alternatively, the multispecific polypeptide construct and
additional agent are separate from each other, e.g., each is
formulated into a separate therapeutic composition, and the
multispecific polypeptide construct and the additional agent are
administered simultaneously, or the multispecific polypeptide
construct and the additional agent are administered at different
times during a treatment regimen. For example, the multispecific
polypeptide construct is administered prior to the administration
of the additional agent, the multispecific polypeptide construct is
administered subsequent to the administration of the additional
agent, or the multispecific polypeptide construct and the
additional agent are administered in an alternating fashion. As
described herein, the multispecific polypeptide construct and
additional agent are administered in single doses or in multiple
doses.
[0112] In some embodiments, the multispecific polypeptide construct
and the additional agent(s) are administered simultaneously. For
example, the multispecific polypeptide construct and the additional
agent(s) can be formulated in a single composition or administered
as two or more separate compositions. In some embodiments, the
multispecific polypeptide construct and the additional agent(s) are
administered sequentially, or the multispecific polypeptide
construct and the additional agent are administered at different
times during a treatment regimen.
[0113] In addition to the elements described above, the
multispecific polypeptide construct can contain additional elements
such as, for example, amino acid sequence N- or C-terminal of the
multispecific polypeptide construct. For example, a multispecific
polypeptide construct can include a targeting moiety to facilitate
delivery to a cell or tissue of interest. Multispecific polypeptide
construct can be conjugated to an agent, such as a therapeutic
agent, a detectable moiety or a diagnostic agent. Examples of
agents are disclosed herein.
[0114] The multispecific polypeptide construct can also include any
of the conjugated agents, linkers and other components described
herein in conjunction with a multispecific polypeptide construct of
the disclosure.
[0115] The disclosure also pertains to immunoconjugates comprising
a multispecific polypeptide construct conjugated to a cytotoxic
agent such as a toxin (e.g., an enzymatically active toxin of
bacterial, fungal, plant, or animal origin, or fragments thereof),
or a radioactive isotope (i.e., a radioconjugate). Suitable
cytotoxic agents for use in targeting diseased T cells such as in a
T cell-derived lymphoma include, for example, dolastatins and
derivatives thereof (e.g. auristatin E, AFP, MMAD, MMAF, MMAE). In
some embodiments, the agent is a dolastatin. In some embodiments,
the agent is an auristatin or derivative thereof. In some
embodiments, the agent is a maytansinoid or maytansinoid
derivative. In some embodiments, the agent is DM1 or DM4. In some
embodiments, the agent is a duocarmycin or derivative thereof. In
some embodiments, the agent is a calicheamicin or derivative
thereof. In some embodiments, the agent is a
pyrrolobenzodiazepine.
[0116] In some embodiments, the linker between the multispecific
polypeptide construct and the cytotoxic agent is cleavable. In some
embodiments, the linker is non-cleavable. In some embodiments, two
or more linkers are present. The two or more linkers are all the
same, e.g., cleavable or non-cleavable, or the two or more linkers
are different, e.g., at least one cleavable and at least one
non-cleavable.
[0117] The multispecific polypeptide constructs and conjugates
thereof are useful in methods for treating a variety of disorders
and/or diseases. Non-limiting examples of disease include: all
types of cancers (breast, lung, colorectal, prostate, melanomas,
head and neck, pancreatic, etc.), rheumatoid arthritis, Crohn's
disuse, SLE, cardiovascular damage, ischemia, etc. For example,
indications would include leukemias, including T-cell acute
lymphoblastic leukemia (T-ALL), lymphoblastic diseases including
multiple myeloma, and solid tumors, including lung, colorectal,
prostate, pancreatic, and breast, including triple negative breast
cancer. For example, indications include bone disease or metastasis
in cancer, regardless of primary tumor origin; breast cancer,
including by way of non-limiting example, ER/PR+ breast cancer,
Her2+ breast cancer, triple-negative breast cancer; colorectal
cancer; endometrial cancer; gastric cancer; glioblastoma; head and
neck cancer, such as esophageal cancer; lung cancer, such as by way
of non-limiting example, non-small cell lung cancer; multiple
myeloma ovarian cancer; pancreatic cancer; prostate cancer;
sarcoma, such as osteosarcoma; renal cancer, such as by way of
nonlimiting example, renal cell carcinoma; and/or skin cancer, such
as by way of nonlimiting example, squamous cell cancer, basal cell
carcinoma, or melanoma. In some embodiments, the cancer is a
squamous cell cancer. In some embodiments, the cancer is a skin
squamous cell carcinoma. In some embodiments, the cancer is an
esophageal squamous cell carcinoma. In some embodiments, the cancer
is a head and neck squamous cell carcinoma. In some embodiments,
the cancer is a lung squamous cell carcinoma.
[0118] Also provided is a pharmaceutical composition comprising any
of the multispecific polypeptide constructs provided herein and a
pharmaceutically acceptable carrier. In some cases, the
pharmaceutical composition is sterile. Pharmaceutical compositions
according to the disclosure can include a multispecific polypeptide
construct of the disclosure and a carrier. These pharmaceutical
compositions can be included in kits, such as, for example,
diagnostic kits.
[0119] One skilled in the art will appreciate that the antibodies
of the disclosure have a variety of uses. For example, the proteins
of the disclosure are used as therapeutic agents for a variety of
disorders. The antibodies of the disclosure are also used as
reagents in diagnostic kits or as diagnostic tools, or these
antibodies can be used in competition assays to generate
therapeutic reagents.
BRIEF DESCRIPTION OF DRAWINGS
[0120] FIG. 1 is a schematic of the basic components of the
multispecific polypeptide constructs of the present disclosure
having constrained CD3 binding. The antigen binding domain(s) are
positioned at the amino and/or carboxy termini. The Fc region, such
as a heterodimeric Fc region, is positioned N-terminal to the CD3
binding region. This positioning of the Fc in close proximity to
the CD3 binding region obstructs CD3 binding. The linker can be a
non-cleavable linker as provided herein.
[0121] FIGS. 2A and B is a schematic of various FRa-targeting
constrained CD3 constructs composed of two polypeptides, Chain 1
and Chain 2. As shown in FIG. 2A, Chain 1 contains a FRa sdAb
(antigen binding domain), linked to a heterodimeric Fc "hole,"
linked via a non-cleavable linker (ranging from 3 amino acids in
cx1356 to 18 amino acids in cx681) to anti-CD3 VL domain, linked to
a second FR.alpha. sdAb; and Chain 2 contains a FR.alpha. sdAb,
linked to a complementary heterodimeric Fc "knob", linked via the
same non-cleavable linker as above to anti-CD3 VH domain, linked to
second FR.alpha. sdAb. When co-expressed the CD3 binding domain is
properly assembled via the association of the VL:VH on the hole and
knob, respectively (FIG. 2B). An exemplary anti-CD3 is a
disulfide-stabilized Fv (dsFv) containing a variable light (VL)
chain with the mutation G100C and a variable heavy (VH) chain with
a mutation G44C.
[0122] FIG. 3A is a schematic of various B7H3-targeting constrained
CD3 constructs composed of two polypeptides, Chain 1 and Chain 2.
Chain 1 contains either a heterodimeric Fc "hole," linked via a
non-cleavable linker to an anti-CD3 VL domain modified at G100C
(top); a B7H3-targeting sdAb linked to a heterodimeric Fc "hole,"
linked via a non-cleavable linker to an anti-CD3 VL domain
(middle); or an B7H3-targeting sdAb linked to a heterodimeric Fc
"hole," linked via a non-cleavable linker to an anti-CD3 VL domain
modified at G100C (bottom). Chain 2 contains either a B7H3-targeted
sdAb, linked to a complementary heterodimeric Fc "knob," linked via
the linker as above to an anti-CD3 VH domain modified at G44C
linked to second B7H3 sdAb (top); a B7H3-targeted sdAb, linked to a
complementary heterodimeric Fc "knob," linked via the linker as
above to an anti-CD3 VH domain (middle); or a B7H3-targeted sdAb,
linked to a complementary heterodimeric Fc "knob," linked via the
linker as above to an anti-CD3 VH domain modified by G44C (bottom).
When co-expressed the CD3 binding domain is properly assembled via
the association of the VL:VH on the hole and knob, respectively.
Where denoted the VH:VL interaction is stabilized by an engineered
disulfide bond between the modified residues G44C in the VH domain
and G100C in the VL domain.
[0123] FIG. 3B is a schematic of various B7H3-targeting constrained
CD3 constructs composed of two polypeptides, Chain 1 and Chain 2.
Chain 1 contains a heterodimeric Fc "hole," linked via a
non-cleavable linker to an anti-CD3 VL domain modified at G100C
linked to a co-stimulatory receptor targeting sdAb. Chain 2
contains either a B7H3-targeted sdAb, linked to a complementary
heterodimeric Fc "knob," linked via the linker as above to an
anti-CD3 VH domain modified at G44C linked to second B7H3-targeted
sdAb (top); a heterodimeric Fc "knob," linked via the linker as
above to an anti-CD3 VH domain modified at G44C linked to a
B7H3-targeted sdAb (middle); or a B7H3-targeted sdAb, linked to a
complementary heterodimeric Fc "knob," linked via the linker as
above to an anti-CD3 VH domain modified by G44C (bottom). When
co-expressed the CD3 binding domain is properly assembled via the
association of the VL:VH on the hole and knob, respectively. VH:VL
interaction is stabilized by an engineered disulfide bond between
the modified residues G44C in the VH domain and G100C in the VL
domain. The resulting constructs are engage B7H3 either in bivalent
(top) or monovalent (middle and bottom) manner. All the constructs
herein express contain a co-stimulatory receptor targeting
sdAb.
[0124] FIG. 3C is a schematic of various B7H3-targeting constrained
CD3 constructs composed of three polypeptides, Chain 1, Chain 2 and
Chain 3, wherein the B7H3 targeting domain is a FAB. Chain 1
contains a BH73-targeting VH, an IgG Constant Heavy 1 (CH1) linked
via a hinge to a first member of a heterodimeric Fc (Fc-Het-1),
linked via the linker as above to an anti-CD3 VL domain that either
lacks (top) or contains the modification of G100C (bottom). Chain 2
contains a BH73-targeting VH, an IgG Constant Heavy 1 (CH1) linked
via a hinge to a second member of a heterodimeric Fc (Fc-Het-2),
linked via the linker as above to an anti-CD3 VH domain that either
lacks (top) or contains the modification of G44C (bottom). Chain 3
contains a complementary B7H3-targeting VL domain linked to human
Ig Constant Light (CL) region. When co-expressed the CD3 binding
domain is properly assembled via the association of the VL:VH on
the complimentary heterodimeric Fc regions. Where denoted the VH:VL
interaction is stabilized by an engineered disulfide bond between
the modified residues G44C in the VH domain and G100C in the VL
domain.
[0125] FIG. 4A is a schematic of various DLL3-targeting constrained
CD3 constructs composed of two polypeptides, Chain 1 and Chain 2.
Chain 1 contains a heterodimeric Fc "hole", linked via a
non-cleavable linker to an anti-CD3 VL domain modified at G100C
linked to a co-stimulatory receptor targeting sdAb. Chain 2
contains either a DLL3-targeted sdAb, linked to a complementary
heterodimeric Fc "knob", linked via the linker as above to an
anti-CD3 VH domain modified at G44C linked to second DLL3-targeted
sdAb (top); a heterodimeric Fc "knob", linked via the linker as
above to an anti-CD3 VH domain modified at G44C linked to a
DLL3-targeted sdAb (middle); or a DLL3-targeted sdAb, linked to a
complementary heterodimeric Fc "knob", linked via the linker as
above to an anti-CD3 VH domain modified by G44C (bottom). When
co-expressed the CD3 binding domain is properly assembled via the
association of the VL:VH on the hole and knob, respectively. VH:VL
interaction is stabilized by an engineered disulfide bond between
the modified residues G44C in the VH domain and G100C in the VL
domain. The resulting constructs are engage DLL3 either in bivalent
(top) or monovalent (middle and bottom) manner. All the constructs
herein express contain a co-stimulatory receptor targeting
sdAb.
[0126] FIG. 4B is a schematic of a DLL3-targeting constrained CD3
construct, cx5499 composed of two polypeptides, Chain 1 and Chain
2. cx5499 is identical to cx5352 shown in FIG. 4A (top), but
lacking co-stimulatory receptor targeting sdAb on the C-terminus of
Chain 1. When co-expressed the CD3 binding domain is properly
assembled via the association of the VL:VH on the hole and knob,
respectively. VH:VL interaction is stabilized by an engineered
disulfide bond between the modified residues G44C in the VH domain
and G100C in the VL domain. The resulting constructs are engage
DLL3 either in bivalent (top) or monovalent (middle and bottom)
manner.
[0127] FIG. 5A-D depict cellular binding by representative
FRa-targeting constrained CD3 engaging constructs, cx1356 and
cx681. FIG. 5A and FIG. 5C show binding to Ovcar5 cells (a
FR.alpha. positive ovarian cancer cell line). FIG. 5B and FIG. 5D
depict the lack of binding to T-cells. FIG. 5A and FIG. 5B display
histograms of the normalized cell counts vs fluorescence at 100 nM
of each construct. The secondary anti-human APC antibody only
control is shown in the filled black trace, while the positive
control anti-CD3 binding is shown in the open trace, and cx1356 and
cx681 are shown in the gray shaded traces in FIG. 5A and FIG. 5B.
The full titration of each construct on the various cell types are
shown in FIG. 5C and FIG. 5D.
[0128] FIG. 6A-F depict cellular binding by representative
B7H3-targeting constrained CD3 engaging constructs. FIGS. 6A, C,
and E show binding to A375 cells (a B7H3 positive human melanoma
cell line). FIGS. 6B, D, and F show the lack of binding to isolated
T cells.
[0129] FIG. 7A-F depict the impact of linker length on the capacity
to agonize CD3 in the presence of FR.alpha. positive IGROV1 cells
(FIG. 7A, 7C, 7E), or FR.alpha. negative NCI-H460 cells (FIG. 7B,
7D, 7F). FIG. 7A-B show the kinetics of CD3 signaling by 2 nM of
various constructs on antigen positive and negative cells,
respectively. FIG. 7C-D show the magnitude of CD3 agonizing
capacity by 2 nM of various constructs on antigen positive and
negative cells, respectively. FIG. 7E-F show the potency of CD3
agonizing capacity of various constructs with differing linker
lengths on antigen positive and negative cells, respectively. A
Jurkat CD3 NFAT-GFP reporter cell line was used to assess CD3
signaling. Constrained CD3 binding proteins only effectively engage
and cluster CD3 on T cells when bound to a second antigen on target
cells.
[0130] FIG. 8A-D depict the ability of representative
B7H3-targeting constrained CD3 engaging constructs to agonize CD3
in a target dependent manner. FIG. 8A and FIG. 8C depict the
capacity to mediate CD3 signaling in the presence of B7H3 positive
A375 cells, while FIG. 8B and FIG. 8D show the inability to mediate
CD3 signaling in the presence of B7H3 negative CCRF-CEM cells. A
Jurkat CD3 NFAT-GFP reporter cell line was used to assess CD3
agonism.
[0131] FIG. 9A depicts the ability of a representative
B7H3-targeting constrained CD3 engaging construct (cx3072) to
induce T-cell mediated cytotoxicity in a target dependent manner.
Target cells were labeled with cytoID red label and dying cells
were visualized by addition of Caspase 3/7 green reagent.
Cytotoxicity was assessed by determining the overlap area of red
target cells and green dying cells. A B7H3 negative A375 cell line,
generated by CRISPR technology was used to test antigen specific
T-cell mediated cytotoxicity. cx3072 was unable to elicit T-cell
mediated cytotoxicity of these B7H3 deficient cells.
[0132] FIG. 9B shows the ability of cx5952 to induce T-cell
mediated cytotoxicity in the presence of B7H3 positive A375 cells,
but not in the presence of B7H3 negative CCRF-CEM cells.
[0133] FIGS. 10A and 10B depict the ability of representative
B7H3-targeting constrained CD3 engaging constructs to induce T-cell
mediated cytotoxicity in a target dependent manner. FIG. 10A
depicts the capacity of these constructs to induce T-cell mediated
cytotoxicity in the presence of B7H3 positive A375 cells, while
FIG. 10B depicts the capacity of these constructs to induce T-cell
mediated cytotoxicity in the presence of B7H3 negative CCRF-CEM
cells. Cytotoxicity was assessed by determining the overlap area of
red target cells and green dying cells.
[0134] FIGS. 10C and 10D depict the ability of representative
B7H3-targeting constrained CD3 engaging constructs to induce T-cell
mediated cytotoxicity in a target dependent manner. FIG. 10C
depicts the capacity of these constructs to induce T-cell mediated
cytotoxicity in the presence of B7H3 positive A375 cells, while
FIG. 10C depicts the capacity of these constructs to induce T-cell
mediated cytotoxicity in the presence of B7H3 negative CCRF-CEM
cells. Cytotoxicity was assessed by determining the overlap area of
red target cells and green dying cells.
[0135] FIGS. 11A-C depict the ability of a representative
B7H3-targeting constrained CD3 engaging construct (cx5952) to
induce T-cell mediated T-cell activation in a target dependent
manner. T-cell activation of CD4+ or CD8+ T cells was assessed by
expression of the T cell activation markers CD25 (FIG. 11A), CD69
(FIG. 11B), and CD71 (FIG. 11C).
[0136] FIGS. 11D-11K depict the ability of representative
B7H3-targeting constrained CD3 engaging constructs to induce T-cell
activation in a target dependent manner. B7H3-target-dependent CD4+
T-cell activation is shown by expression of the T cell activation
markers CD25 (FIG. 11D) and CD71 (FIG. 11F). B7H3-target-dependent
CD8+ T-cell activation is shown by expression of the T cell
activation markers CD25 (FIG. 11H) and CD71 (FIG. 11J). T-cell
activation was not observed in the absence of B7H3 positive cells,
based on T cell activation marker CD25 as shown in CD4+ T cells
(FIG. 11E) or CD8+ T cells (FIG. 11I) or based on T cell activation
marker CD71 as shown in CD4+ T cells (FIG. 11G) or CD8+ T cells
(FIG. 11K)
[0137] FIG. 12A depicts the ability of representative
B7H3-targeting constrained CD3 engaging constructs to induce
IFN.gamma. production in a target dependent manner. FIG. 12A shows
the production of IFN.gamma. from T-cells cultured with B7H3
positive A375 cells and in the presence of B7H3 negative CCRF-CEM
cells in the presence of the representative B7H3-targeting CD3
engaging constructs.
[0138] FIG. 12B depicts the ability of representative
B7H3-targeting constrained CD3 engaging constructs to induce
IFN.gamma. production in a target dependent manner. FIG. 12B shows
the production of IFN.gamma. from T-cells cultured with B7H3
positive A375 cells and in the presence of B7H3 negative CCRF-CEM
cells in the presence of the representative B7H3-targeting CD3
engaging constructs.
[0139] FIGS. 13A and 13B depict cellular binding of representative
B7H3-targeting constrained CD3 engaging constructs. cx5187 and
cx5823 each contain two B7H3 binding domains while complex cx5873
and cx5965 each contain one B7H3 binding domain. FIG. 13A shows
binding to B7H3 positive A375 cells. FIG. 13B shows the lack of
binding to B7H3 negative CCRF-CEM cells and isolated T-cells.
[0140] FIG. 13C and FIG. 13D depict the ability of representative
B7H3-targeting constrained CD3 engaging constructs to agonize CD3
in a target dependent manner. FIG. 13C shows that engaging B7H3
positive A375 cells with a construct hat is bivalent and
bi-epitopic to B7H3 (cx5187) induced more potent CD3 signaling than
constructs that are monovalent to B7H3 (cx5873 and cx5965). FIG.
13D shows the lack of activation of T-cells in the presence of B7H3
negative CCRF-CEM cells. A Jurkat CD3 NFAT-GFP reporter cell line
was used to assess CD3 agonism.
[0141] FIG. 14A and FIG. 14B depict the ability of representative
B7H3-targeting constrained CD3 engaging constructs to induce T-cell
mediated cytotoxicity in a target dependent manner. FIG. 14A shows
that targeting B7H3 positive A375 cells with a construct that is
bivalent and bi-epitopic to B7H3 (cx5187) induced more potent
T-cell mediated cytotoxicity than constructs that are monovalent to
B7H3 (cx5873 and cx5965). FIG. 14B depicts the lack of T-cell
mediated cytotoxicity against B7H3 negative CCRF-CEM cells.
[0142] FIG. 15A-D depict the ability of representative
B7H3-targeting constrained CD3 engaging molecules to activate
T-cells in the presence of B7H3 positive A375cells, but not in the
presence of B7H3 negative CCRF-CEM cells. FIGS. 15A and 15B show
that targeting B7H3 positive A375 cells with a construct that is
bivalent and bi-epitopic to B7H3 (cx5187), induced more potent CD25
expression on CD4+ and CD8+ T-cells than constructs that are
monovalent to B7H3 (cx5873 and cx5965). FIGS. 15C and 15D show the
lack of CD25 expression on CD4+ and CD8+ T-cells in the presence of
B7H3 negative CCRF-CEM cells.
[0143] FIGS. 16A and 16B demonstrate the ability of representative
B7H3-targeting constrained CD3 engaging constructs to elicit T-cell
mediated cytotoxicity in the presence of B7H3-positive A375 cells
(FIG. 16A) but not in the presence of CCRF-CEM B7H3-negative cells
(FIG. 16B).
[0144] FIGS. 16C-16J demonstrate the ability of representative
B7H3-targeting constrained CD3 engaging constructs to elicit T cell
activation in the presence of B7H3-positive A375 cells but not in
the presence of CCRF-CEM B7H3-negative cells, as assessed by:
expression of CD25 on CD4+ T cells (FIGS. 16C and 16D,
respectively), CD25 expression on CD8+ T cells (FIGS. 16E and 16F,
respectively), CD71 expression on CD4+ T cells (FIGS. 16G and 1611,
respectively), CD71 expression on CD8+ T cells (FIGS. 161 and 16J,
respectively).
[0145] FIGS. 16K and 16L demonstrate the ability of representative
B7H3-targeting constrained CD3 engaging constructs to elicit T cell
cytokine production in the presence of B7H3-positive A375 cells
(FIG. 16K) but not in the presence of CCRF-CEM B7H3-negative cells
(FIG. 16L).
[0146] FIGS. 17A and 17B demonstrate that representative monovalent
(cx5800 and cx5801) and bivalent (cx5352) DLL3-targeting
constrained CD3 engaging constructs bound to a DLL3 expressing cell
line, SHP-77 (FIG. 17A), but not to isolated T-cells (FIG. 17B).
Binding was assessed by flow cytometry.
[0147] FIG. 17C depicts the ability of representative
DLL3-targeting constrained CD3 engaging constructs to agonize CD3
signaling in the presence of DLL3 positive SHP-77 cells. Engaging
DLL3 positive cells with a construct that is bivalent and
bi-epitopic to DLL3 (cx5352) induced more potent T-cell activation
than constructs that are monovalent to DLL3 (cx5800 and cx5801). A
Jurkat CD3 NFAT-Luciferase reporter cell line was used to assess
CD3 signaling.
[0148] FIG. 18A-18E demonstrate the ability of a representative
DLL3-targeting constrained CD3 engaging construct, cx5499 to elicit
T-cell mediated cytotoxicity and T-cell activation in the presence
of DLL3-positive SHP-77 cells. FIG. 18A demonstrates the ability of
the representative DLL3-targeting constrained CD3 engaging
constructs to elicit T-cell mediated cytotoxicity in the presence
of DLL3-positive SHP-77 cells. FIGS. 18C-18D demonstrate the
ability of representative DLL3-targeting constrained CD3 engaging
constructs to elicit T cell activation in the presence of
DLL3-positive SHP-77 cells, as assessed by: expression of CD25 on
CD4+ T cells (FIG. 18B), CD69 expression on CD4+ T cells (FIG.
18C), CD25 expression on CD8+ T cells (FIG. 18D) and CD69
expression on CD8+ T cells (FIG. 18E).
DETAILED DESCRIPTION
[0149] The present disclosure provides constrained T-cell engaging
fusion proteins in the form of multispecific polypeptide constructs
that bind at least CD3 and a second antigen. The multispecific
polypeptide constructs provided herein include at least a first
component that includes one or more copies of an antigen-binding
domain that binds an antigen operably linked to an immunoglobulin
Fc region, a second component that includes one or more copies of
at least a binding domain that binds CD3 (referred to herein as an
anti-CD3 binding domain or a CD3 binding region, which are terms
that are used interchangeably herein), and a linker, such as a
polypeptide linker, that joins the first component and the second
component. In some embodiments, the antigen is a tumor associated
antigen (TAA). In some embodiments, the linker is a non-cleavable
linker. In some embodiments, the linker does not contain a
substrate recognition site that is specifically recognized by a
protease, such as a protease that is granzyme B, an MMP or
matriptase.
[0150] The provided multispecific polypeptide constructs include a
configuration in which the first component containing the Fc region
is N-terminal to the second component containing the CD3 binding
region. In such an embodiment, the first and second components are
joined via a linker that is C-terminal to the end of the Fc region.
In some embodiments the antigen binding domain(s) is positioned on
the amino-terminal (N-term) region of the multispecific polypeptide
construct. In some embodiments, the antigen binding domain(s) is
positioned on the carboxy-terminal (C-term) region of the
multispecific polypeptide construct. In some embodiments, the
antigen binding domain(s) is positioned on both the N- and
C-terminal regions of the multispecific polypeptide construct.
Various configurations of a multispecific polypeptide construct as
provided herein are shown in FIG. 1.
[0151] The provided multispecific polypeptide constructs exhibit
constrained T-cell engaging activity because such constructs only
substantially bind to CD3 once an antigen is bound via the
antigen-binding domain. This is exemplified in the Examples and
Figures provided herein, which demonstrate the ability of
constrained CD3-engaging proteins to efficiently bind TAA positive
cells, while having little to no binding of T cells. This unique
property allows constrained CD3-engaging proteins to distribute to
sites where TAA is present without binding to peripheral T cells.
This format is distinct from other CD3 engaging multispecific
constructs, in that constitutive CD3 binding is disallowed or
eliminated, providing a significant benefit by avoiding peripheral
T-cell binding and permitting superior distribution to the site(s)
where antigen is present as recognized by the antigen binding
domain. Furthermore, other CD3 engaging constructs mediate
antigen-dependent T-cell activation. However, the multispecific
polypeptide constructs provided herein mediate both antigen
dependent T-cell binding and activation.
[0152] The constrained T-cell engaging activity of the provided
multispecific polypeptide constructs is due, in some aspects, to
the positioning of the Fc region N-terminal to the CD3-binding
region. In some embodiments, such positioning reduces, attenuates,
dampens and/or prevents CD3 binding by the CD3 binding region. In
the absence of antigen binding by the antigen binding domain, the
multispecific polypeptide constructs provided herein demonstrate
reduced or eliminated CD3 binding and T-cell activating capacity.
In some embodiments, in the presence of an antigen binding event
mediated by the antigen binding domain(s) of the multispecific
polypeptide constructs, the capacity to bind CD3 by the CD3 binding
region is greatly enhanced. In some embodiments, in the presence of
an antigen binding event mediated by the antigen binding domains(s)
of the multispecific polypeptide constructs, the capacity to
activate T-cells is greatly enhanced. Engagement of its cognate
antigen by the antigen binding domain(s) within the multispecific
polypeptide construct leads to subsequent T-cell engagement and
mediates antigen-dependent T-cell activation, such as cytotoxicity,
cytokine release, degranulation and proliferation. In some
embodiments, the provided multispecific polypeptide constructs can
be used to increase an immune response, such as to enhance T-cell
activity, including cytolytic (or cytotoxic) T-cell activity. The
modulation of the immune response can, in some aspects, treat a
disease or condition in a subject.
[0153] In some embodiments, the one or more antigen binding domains
bind an antigen on a tumor cell or a cell of the tumor
microenvironment. In some aspects, the provided multispecific
polypeptide constructs can be used to increase immune responses,
such as T-cell activity, e.g. cytotoxicity activity, against a
tumor or cancer. In some embodiments, the provided multispecific
polypeptide constructs can be used to treat a tumor or cancer in
the subject.
[0154] In some embodiments, the multispecific polypeptide
constructs of the disclosure ensure that there will be no binding
of T-cells via CD3 in peripheral blood, as the CD3 binding region
of these constructs is constrained or otherwise blocked and/or
inhibited by the presence of the Fc region. Thus, the multispecific
polypeptide constructs of the disclosure provide a number of
advantages. In some aspects, these constructs limit the sink effect
caused by binding all T-cells. In some aspects, these constructs
reduce systemic toxicity.
[0155] In some embodiments, the provided multispecific polypeptide
constructs of the disclosure allow for controlled biodistribution
to a desired site in a subject, such as, for example, a site of
tumor-associated antigen (TAA) expression. Sites of TAA expression
include, for example, tumor and the surrounding tumor
microenvironment.
[0156] In some embodiments, the multispecific polypeptide
constructs of the disclosure exhibit specificity for CD3 and one or
more other antigen. In some embodiments, the multispecific
polypeptide constructs can contain more than one antigen binding
domain able to bind one or more TAA, such as 2, 3 or 4 antigen
binding domains, see e.g. FIG. 1. In some embodiments, the one or
more antigen binding domains bind the same antigen. In some
embodiments, the multispecific polypeptide constructs include more
than one antigen binding domains that bind distinct epitopes on the
same antigen. In some embodiments, the multispecific polypeptide
constructs include more than one antigen binding domains that bind
one or more distinct antigens. In some embodiments, the
multispecific polypeptide constructs include more than one antigen
binding domains that bind distinct epitopes on the same antigens as
well as include additional antigen binding domains that bind to one
or more distinct antigens. In some aspects, the provided
multispecific polypeptide constructs are bispecific polypeptide
constructs, such that they are able to bind to CD3 and to another
antigen, such as a TAA, via binding of the antigen-binding domain
of the multispecific polypeptide construct. In some examples, the
provided multispecific polypeptide constructs are bispecific
polypeptide constructs that provide multivalent engagement of one
or more TAA through the use of a first antigen-binding domain and a
second antigen-binding domain. For example, in some embodiments,
the bispecific polypeptide constructions include a first
antigen-binding single domain antibody (sdAb) and a second
antigen-binding sdAb.
[0157] In some embodiments, the multispecific polypeptide
constructs provided herein exist in two states in terms of capacity
to bind CD3 and subsequently activate T-cells: (1) the "inactive"
state occurs when there is no binding of any or all of the antigen
binding domain(s), such that the CD3 binding is constrained and
T-cell interaction is obviated, and (2) the "active" state occurs
upon antigen binding by any or all of the antigen binding
domain(s), such that the CD3 binding region is able to bind CD3 and
the T-cell interaction is allowed.
[0158] In some embodiments, the Fc region is linked to the CD3
binding domain via a linker or linkers. In some embodiments, the Fc
region is linked to the CD3 binding region via a non-cleavable
linker or linkers, such as any as described.
[0159] In some embodiments, the Fc region is a homodimeric Fc
region. In some embodiments, the Fc region is a heterodimeric Fc
region. In some embodiments, the Fc region is a monomeric Fc
region. In some embodiments, the Fc region of the multispecific
polypeptide constructs are capable of interacting with Fc.gamma.Rs
and mediating innate immune effector functions, for example,
antibody dependent cellular toxicity (ADCC) and antibody dependent
cellular phagocytosis (ADCP). In some embodiments, the Fc region of
the multispecific polypeptide constructs are capable of interacting
with complement proteins, namely C1q, and mediating complement
dependent cytotoxicity. Thus, in some aspects, the multispecific
polypeptide constructs of the disclosure allow for multiple immune
effector mechanisms, including innate immune effectors and
T-cells.
[0160] In some embodiments, the multispecific polypeptide
constructs of the disclosure allow for T-cell and NK cell mediated
cytotoxicity to occur simultaneously. In some cases, such activity
can occur in a multispecific polypeptide construct in which is
contained a first antigen binding domain, e.g., a first anti-TAA
antigen binding domain, and a second antigen binding domain, e.g.,
a second anti-TAA antigen binding domain, that can target distinct
and/or non-competing epitopes on a given TAA.
[0161] It is contemplated that the constrained CD3 engaging
constructs are amenable for use with any TAA-binding domain,
allowing better therapeutic exposure within the tumor or
tumor-microenvironment by avoiding interactions with peripheral
T-cells and mediating potent TAA-dependent T-cell cytotoxicity. In
some embodiments, the second portion or component contains a CD3
binding region that is monovalent to CD3, such that there will be
no activation of T-cell unless there is TAA present.
[0162] In some aspects, the multispecific polypeptide constructs of
the disclosure provide a number of advantages over current
bispecific therapeutics. The multispecific polypeptide constructs
of the disclosure are smaller than a conventional therapeutic
antibody, e.g., 150 kDa vs. 125 kDa, which will allow for better
target, e.g. tumor, penetration. In some aspects, the size of the
entire multispecific polypeptide construct provides long half-life
for the construct. In some aspects, the multispecific polypeptide
constructs of the disclosure exhibit reduced systemic toxicity or
toxicity of any area outside the tumor and/or tumor
microenvironment, since CD3 binding by the CD3 binding region
depends on TAA engagement before CD3 engagement will occur.
[0163] All publications and patent documents cited herein are
incorporated herein by reference as if each such publication or
document was specifically and individually indicated to be
incorporated herein by reference. Citation of publications and
patent documents is not intended as an admission that any is
pertinent prior art, nor does it constitute any admission as to the
contents or date of the same. The invention having now been
described by way of written description, those of skill in the art
will recognize that the invention can be practiced in a variety of
embodiments and that the foregoing description and examples below
are for purposes of illustration and not limitation of the claims
that follow.
I. DEFINITIONS
[0164] Unless otherwise defined, scientific and technical terms
used in connection with the present disclosure shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The term "a" entity or "an" entity refers to one or more
of that entity. For example, a compound refers to one or more
compounds. As such, the terms "a", "an", "one or more" and "at
least one" can be used interchangeably. Further, unless otherwise
required by context, singular terms shall include pluralities and
plural terms shall include the singular. Generally, nomenclatures
utilized in connection with, and techniques of, cell and tissue
culture, molecular biology, and protein and oligo- or
polynucleotide chemistry and hybridization described herein are
those well-known and commonly used in the art. Standard techniques
are used for recombinant DNA, oligonucleotide synthesis, and tissue
culture and transformation (e.g., electroporation, lipofection).
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications or as commonly
accomplished in the art or as described herein. The foregoing
techniques and procedures are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the present specification. See e.g., Sambrook
et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The
nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well-known and commonly used in the art.
Standard techniques are used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery,
and treatment of patients.
[0165] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0166] As used herein, the term "antibody" refers to immunoglobulin
molecules and antigen-binding portions of immunoglobulin (Ig)
molecules, i.e., molecules that contain an antigen binding site
that specifically binds (immunoreacts with) an antigen. By
"specifically bind" or "immunoreacts with" or "immunospecifically
bind" is meant that the antibody reacts with one or more antigenic
determinants of the desired antigen and does not react with other
polypeptides or binds at much lower affinity
(K.sub.d>10.sup.-6). Antibodies include, but are not limited to,
polyclonal, monoclonal, chimeric, fully human, domain antibody,
single chain, Fab, and F(ab').sub.2 fragments, Fvs, scFvs, and a
Fab expression library. Typically, an "antigen-binding fragment"
contains at least one CDR of an immunoglobulin heavy and/or light
chain that binds to at least one epitope of the antigen of
interest. In this regard, an antigen-binding fragment may comprise
1, 2, 3, 4, 5, or all 6 CDRs of a variable heavy chain (VH) and
variable light chain (VL) sequence from antibodies that bind the
antigen, such as generally six CDRs for an antibody containing a VH
and a VL ("CDR1," "CDR2" and "CDR3" for each of a heavy and light
chain), or three CDRs for an antibody containing a single variable
domain. Antigen binding fragments include single domain antibodies,
such as those only containing a VH or only containing a VL,
including, for example, V.sub.HH, V.sub.NAR, engineered V.sub.H or
V.sub.K domains.
[0167] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. In general, antibody
molecules obtained from humans relate to any of the classes IgG,
IgM, IgA, IgE and IgD, which differ from one another by the nature
of the heavy chain present in the molecule. Certain classes have
subclasses as well, such as IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, and others. Furthermore, in humans, the light chain may
be a kappa chain or a lambda chain.
[0168] The term "monoclonal antibody" (mAb) or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one molecular species of antibody
molecule consisting of a unique light chain gene product and a
unique heavy chain gene product. In particular, the complementarity
determining regions (CDRs) of the monoclonal antibody are identical
in all the molecules of the population. MAbs contain an antigen
binding site capable of immunoreacting with a particular epitope of
the antigen characterized by a unique binding affinity for it.
[0169] The term "antigen-binding site" or "binding portion" refers
to the part of the immunoglobulin molecule that participates in
antigen binding. The antigen binding site is formed by amino acid
residues of the N-terminal variable ("V") regions of the heavy
("H") and light ("L") chains. Three highly divergent stretches
within the V regions of the heavy and light chains, referred to as
"hypervariable regions," are interposed between more conserved
flanking stretches known as "framework regions," or "FRs". Thus,
the term "FR" refers to amino acid sequences that are naturally
found between, and adjacent to, hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs." The assignment of
amino acids to each domain is in accordance with the definitions of
Kabat Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia
& Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature
342:878-883 (1989).
[0170] As used herein, the term "epitope" includes any specific
portion of an antigen targeted by an antibody, antibody fragment or
other binding domain. The term "epitope" includes any protein
region to which specific binding is directed. The term "epitope"
includes any protein determinant capable of specific binding to an
immunoglobulin or T-cell receptor. Epitopic determinants usually
consist of chemically active surface groupings of molecules such as
amino acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. For example, antibodies may be raised against
N-terminal, central, or C-terminal peptides of a polypeptide. In
addition, antibodies may be raised against linear or discontinuous
epitopes of a polypeptide. An antibody is said to specifically bind
an antigen when the dissociation constant is .ltoreq.1 .mu.M; for
example, in some embodiments .ltoreq.100 nM and in some
embodiments, .ltoreq.10 nM and does not display binding to other
proteins either closely related or distinct.
[0171] As used herein, the terms "specific binding," "immunological
binding," and "immunological binding properties" refer to the
non-covalent interactions of the type that occur between an
immunoglobulin molecule and an antigen for which the immunoglobulin
is specific. The strength, or affinity of immunological binding
interactions can be expressed in terms of the dissociation constant
(K.sub.d) of the interaction, wherein a smaller K.sub.d represents
a greater affinity. Immunological binding properties of selected
polypeptides can be quantified using methods well known in the art.
One such method entails measuring the rates of antigen-binding
site/antigen complex formation and dissociation, wherein those
rates depend on the concentrations of the complex partners, the
affinity of the interaction, and geometric parameters that equally
influence the rate in both directions. Thus, both the "on rate
constant" (K.sub.m) and the "off rate constant" (K.sub.off) can be
determined by calculation of the concentrations and the actual
rates of association and dissociation. (See Nature 361:186-87
(1993)). The ratio of K.sub.off/K.sub.on enables the cancellation
of all parameters not related to affinity and is equal to the
dissociation constant K.sub.d. (See, generally, Davies et al.
(1990) Annual Rev Biochem 59:439-473). An antibody of the present
disclosure is said to specifically bind to EGFR, when the binding
constant (K.sub.d) is .ltoreq.1 .mu.M, for example, in some
embodiments .ltoreq.100 nM, in some embodiments 10 nM, and in some
embodiments .ltoreq.100 .mu.M to about 1 .mu.M, as measured by
assays such as radioligand binding assays or similar assays known
to those skilled in the art.
[0172] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide of genomic, cDNA, or synthetic origin or some
combination thereof, which by virtue of its origin the "isolated
polynucleotide" (1) is not associated with all or a portion of a
polynucleotide in which the "isolated polynucleotide" is found in
nature, (2) is operably linked to a polynucleotide that it is not
linked to in nature, or (3) does not occur in nature as part of a
larger sequence. Polynucleotides in accordance with the disclosure
include the nucleic acid molecules encoding the heavy chain
immunoglobulin molecules shown herein, and nucleic acid molecules
encoding the light chain immunoglobulin molecules shown herein.
[0173] The term "isolated protein" referred to herein means a
protein of cDNA, recombinant RNA, or synthetic origin or some
combination thereof, which by virtue of its origin, or source of
derivation, the "isolated protein" (1) is not associated with
proteins found in nature, (2) is free of other proteins from the
same source, e.g., free of murine proteins, (3) is expressed by a
cell from a different species, or (4) does not occur in nature.
[0174] The term "polypeptide" is used herein as a generic term to
refer to native protein, fragments, or analogs of a polypeptide
sequence. Hence, native protein fragments, and analogs are species
of the polypeptide genus. Polypeptides in accordance with the
disclosure comprise the heavy chain immunoglobulin molecules shown
herein, and the light chain immunoglobulin molecules shown herein,
as well as antibody molecules formed by combinations comprising the
heavy chain immunoglobulin molecules with light chain
immunoglobulin molecules, such as kappa light chain immunoglobulin
molecules, and vice versa, as well as fragments and analogs
thereof.
[0175] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and that has not been intentionally
modified by man in the laboratory or otherwise is
naturally-occurring.
[0176] The term "operably linked" as used herein refers to
positions of components so described are in a relationship
permitting them to function in their intended manner. A control
sequence "operably linked" to a coding sequence is ligated in such
a way that expression of the coding sequence is achieved under
conditions compatible with the control sequences.
[0177] The term "control sequence" as used herein refers to
polynucleotide sequences that are necessary to effect the
expression and processing of coding sequences to which they are
ligated. The nature of such control sequences differs depending
upon the host organism in prokaryotes, such control sequences
generally include promoter, ribosomal binding site, and
transcription termination sequence in eukaryotes, generally, such
control sequences include promoters and transcription termination
sequence. The term "control sequences" is intended to include, at a
minimum, all components whose presence is essential for expression
and processing, and can also include additional components whose
presence is advantageous, for example, leader sequences and fusion
partner sequences. The term "polynucleotide" as referred to herein
means nucleotides of at least 10 bases in length, either
ribonucleotides or deoxynucleotides or a modified form of either
type of nucleotide. The term includes single and double stranded
forms of DNA.
[0178] The term "oligonucleotide" referred to herein includes
naturally occurring, and modified nucleotides linked together by
naturally occurring, and non-naturally occurring oligonucleotide
linkages. Oligonucleotides are a polynucleotide subset generally
comprising a length of 200 bases or fewer. In some embodiments,
oligonucleotides are 10 to 60 bases in length, for example, in some
embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in
length. Oligonucleotides are usually single stranded, e.g., for
probes, although oligonucleotides may be double stranded, e.g., for
use in the construction of a gene mutant. Oligonucleotides of the
disclosure are either sense or antisense oligonucleotides.
[0179] The term "naturally occurring nucleotides" referred to
herein includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" referred to herein includes nucleotides with
modified or substituted sugar groups and the like. The term
"oligonucleotide linkages" referred to herein includes
oligonucleotide linkages such as phosphorothioate,
phosphorodithioate, phosphoroselerloate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoronmidate, and the
like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986);
Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl.
Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539
(1991); Zon et al. Oligonucleotides and Analogues: A Practical
Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press,
Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;
Uhlmann and Peyman Chemical Reviews 90:543 (1990). An
oligonucleotide can include a label for detection, if desired.
[0180] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
Associates, Sunderland? Mass. (1991)). Stereoisomers (e.g., D-amino
acids) of the twenty conventional amino acids, unnatural amino
acids such as .alpha.-, .alpha.-disubstituted amino acids, N-alkyl
amino acids, lactic acid, and other unconventional amino acids may
also be suitable components for polypeptides of the present
disclosure. Examples of unconventional amino acids include: 4
hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and imino acids (e.g., 4-hydroxyproline).
In the polypeptide notation used herein, the left-hand direction is
the amino terminal direction and the right-hand direction is the
carboxy-terminal direction, in accordance with standard usage and
convention.
[0181] Similarly, unless specified otherwise, the left-hand end of
single-stranded polynucleotide sequences is the 5' end the
left-hand direction of double-stranded polynucleotide sequences is
referred to as the 5' direction. The direction of 5' to 3' addition
of nascent RNA transcripts is referred to as the transcription
direction sequence regions on the DNA strand having the same
sequence as the RNA and that are 5' to the 5' end of the RNA
transcript are referred to as "upstream sequences", sequence
regions on the DNA strand having the same sequence as the RNA and
that are 3' to the 3' end of the RNA transcript are referred to as
"downstream sequences".
[0182] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, for example, in some
embodiments, at least 90 percent sequence identity, in some
embodiments, at least 95 percent sequence identity, and in some
embodiments, at least 99 percent sequence identity.
[0183] In some embodiments, residue positions that are not
identical differ by conservative amino acid substitutions.
[0184] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the present disclosure,
providing that the variations in the amino acid sequence maintain
at least 75%, for example, in some embodiments, at least 80%, 90%,
95%, and in some embodiments 99%. In particular, conservative amino
acid replacements are contemplated. Conservative replacements are
those that take place within a family of amino acids that are
related in their side chains. Genetically encoded amino acids are
generally divided into families: (1) acidic amino acids are
aspartate, glutamate; (2) basic amino acids are lysine, arginine,
histidine; (3) non-polar amino acids are alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan, and (4)
uncharged polar amino acids are glycine, asparagine, glutamine,
cysteine, serine, threonine, tyrosine. The hydrophilic amino acids
include arginine, asparagine, aspartate, glutamine, glutamate,
histidine, lysine, serine, and threonine. The hydrophobic amino
acids include alanine, cysteine, isoleucine, leucine, methionine,
phenylalanine, proline, tryptophan, tyrosine and valine. Other
families of amino acids include (i) serine and threonine, which are
the aliphatic-hydroxy family; (ii) asparagine and glutamine, which
are the amide containing family; (iii) alanine, valine, leucine and
isoleucine, which are the aliphatic family; and (iv) phenylalanine,
tryptophan, and tyrosine, which are the aromatic family. For
example, it is reasonable to expect that an isolated replacement of
a leucine with an isoleucine or valine, an aspartate with a
glutamate, a threonine with a serine, or a similar replacement of
an amino acid with a structurally related amino acid will not have
a major effect on the binding or properties of the resulting
molecule, especially if the replacement does not involve an amino
acid within a framework site. Whether an amino acid change results
in a functional peptide can readily be determined by assaying the
specific activity of the polypeptide derivative. Assays are
described in detail herein. Fragments or analogs of antibodies or
immunoglobulin molecules can be readily prepared by those of
ordinary skill in the art. In some embodiments, amino- and
carboxy-termini of fragments or analogs occur near boundaries of
functional domains. Structural and functional domains can be
identified by comparison of the nucleotide and/or amino acid
sequence data to public or proprietary sequence databases.
Computerized comparison methods are used to identify sequence
motifs or predicted protein conformation domains that occur in
other proteins of known structure and/or function. Methods to
identify protein sequences that fold into a known three-dimensional
structure are known. Bowie et al. Science 253:164 (1991). Thus, the
foregoing examples demonstrate that those of skill in the art can
recognize sequence motifs and structural conformations that may be
used to define structural and functional domains in accordance with
the disclosure.
[0185] In some embodiments, amino acid substitutions are those
that: (1) reduce susceptibility to proteolysis, (2) reduce
susceptibility to oxidation, (3) alter binding affinity for forming
protein complexes, (4) alter binding affinities, and (4) confer or
modify other physicochemical or functional properties of such
analogs. Analogs can include various muteins of a sequence other
than the naturally-occurring peptide sequence. For example, single
or multiple amino acid substitutions (for example, conservative
amino acid substitutions) may be made in the naturally-occurring
sequence (for example, in the portion of the polypeptide outside
the domain(s) forming intermolecular contacts. A conservative amino
acid substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles (Creighton, Ed., W.
H. Freeman and Company, New York (1984)); Introduction to Protein
Structure (C. Branden and J. Tooze, eds., Garland Publishing, New
York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).
[0186] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino terminal and/or carboxy-terminal
deletion and/or one or more internal deletion(s), but where the
remaining amino acid sequence is identical to the corresponding
positions in the naturally-occurring sequence deduced, for example,
from a full length cDNA sequence. Fragments typically are at least
5, 6, 8 or 10 amino acids long, for example, in some embodiments,
at least 14 amino acids long, in some embodiments, at least 20
amino acids long, usually at least 50 amino acids long, and in some
embodiments, at least 70 amino acids long. The term "analog" as
used herein refers to polypeptides that are comprised of a segment
of at least 25 amino acids that has substantial identity to a
portion of a deduced amino acid sequence and that has specific
binding to EGFR, under suitable binding conditions. Typically,
polypeptide analogs comprise a conservative amino acid substitution
(or addition or deletion) with respect to the naturally-occurring
sequence. Analogs typically are at least 20 amino acids long, for
example, in some embodiments, at least 50 amino acids long or
longer, and can often be as long as a full-length
naturally-occurring polypeptide.
[0187] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials.
[0188] As used herein, the terms "label" or "labeled" refers to
incorporation of a detectable marker, e.g., by incorporation of a
radiolabeled amino acid or attachment to a polypeptide of biotinyl
moieties that can be detected by marked avidin (e.g., streptavidin
containing a fluorescent marker or enzymatic activity that can be
detected by optical or calorimetric methods). In certain
situations, the label or marker can also be therapeutic. Various
methods of labeling polypeptides and glycoproteins are known in the
art and may be used. Examples of labels for polypeptides include,
but are not limited to, the following: radioisotopes or
radionuclides (e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I), fluorescent
labels (e.g., a fluorophore, rhodamine, lanthanide phosphors),
enzymatic labels (e.g., horseradish peroxidase, p-galactosidase,
luciferase, alkaline phosphatase), chemiluminescent, biotinyl
groups, predetermined polypeptide epitopes recognized by a
secondary reporter (e.g., leucine zipper pair sequences, binding
sites for secondary antibodies, metal binding domains, epitope
tags). In some embodiments, labels are attached by spacer arms of
various lengths to reduce potential steric hindrance. The term
"pharmaceutical agent or drug" as used herein refers to a chemical
compound or composition capable of inducing a desired therapeutic
effect when properly administered to a patient.
[0189] As used herein, a composition refers to any mixture of two
or more products, substances, or compounds, including cells. It may
be a solution, a suspension, liquid, powder, a paste, aqueous,
non-aqueous or any combination thereof.
[0190] The term "pharmaceutical composition" refers to a
composition suitable for pharmaceutical use in a mammalian subject,
often a human. A pharmaceutical composition typically comprises an
effective amount of an active agent (e.g., multispecific
polypeptide construct) and a carrier, excipient, or diluent. The
carrier, excipient, or diluent is typically a pharmaceutically
acceptable carrier, excipient or diluent, respectively.
[0191] The terms "treating," "treatment," or "therapy" of a disease
or disorder as used herein mean slowing, stopping or reversing the
disease or disorders progression, as evidenced by decreasing,
cessation or elimination of either clinical or diagnostic symptoms,
by administration of a pharmaceutical composition of the disclosure
either alone or in combination with another compound as described
herein. "Treating," "treatment," or "therapy" also means a decrease
in the severity of symptoms in an acute or chronic disease or
disorder or a decrease in the relapse rate. As used herein in the
context of cancer, the terms "treatment" or, "inhibit,"
"inhibiting" or "inhibition" of cancer refers to at least one of: a
statistically significant decrease in the rate of tumor growth, a
cessation of tumor growth, or a reduction in the size, mass,
metabolic activity, or volume of the tumor, as measured by standard
criteria such as, but not limited to, the Response Evaluation
Criteria for Solid Tumors (RECIST), or a statistically significant
increase in progression free survival (PFS) or overall survival
(OS). "Preventing," "prophylaxis," or "prevention" of a disease or
disorder refers to administration of a pharmaceutical composition,
either alone or in combination with another compound, to prevent
the occurrence or onset of a disease or disorder or some or all of
the symptoms of a disease or disorder or to lessen the likelihood
of the onset of a disease or disorder.
[0192] The terms "effective amount" or "therapeutically effective
amount" refer to a quantity and/or concentration of a composition
that when administered into a patient either alone (i.e., as a
monotherapy) or in combination with additional therapeutic agents,
yields a statistically significant decrease in disease progression
as, for example, by ameliorating or eliminating symptoms and/or the
cause of the disease. An effective amount may be an amount that
relieves, lessens, or alleviates at least one symptom or biological
response or effect associated with a disease or disorder, prevents
progression of the disease or disorder, or improves physical
functioning of the patient.
[0193] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the
composition), and a substantially purified fraction is a
composition wherein the object species comprises at least about 50
percent (on a molar basis) of all macromolecular species
present.
[0194] Generally, a substantially pure composition will comprise
more than about 80 percent of all macromolecular species present in
the composition, for example, in some embodiments, more than about
85%, 90%, 95%, and 99%. In some embodiments, the object species is
purified to essential homogeneity (contaminant species cannot be
detected in the composition by conventional detection methods)
wherein the composition consists essentially of a single
macromolecular species.
[0195] The term patient includes human and veterinary subjects.
[0196] Other chemistry terms herein are used according to
conventional usage in the art, as exemplified by The McGraw-Hill
Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San
Francisco (1985)).
[0197] The term "about" as used herein refers to the usual error
range for the respective value readily known to the skilled person
in this technical field. Reference to "about" a value or parameter
herein includes (and describes) embodiments that are directed to
that value or parameter per se. For example, description referring
to "about X" includes description of "X".
II. MULTISPECIFIC POLYPEPTIDE CONSTRUCTS
[0198] Provided herein is a multispecific polypeptide construct
containing a first component containing an immunoglobulin Fc region
and a second component comprising a CD3-binding region, wherein the
first and second components are coupled by a linker, wherein the Fc
region is positioned N-terminal to the CD3-binding region; and one
or both of the first and second components comprises an antigen
binding domain that binds a tumor associated antigen (TAA). In some
embodiments, the linker is a non-cleavable linker. In some
embodiments, the linker does not contain a substrate recognition
that is specifically recognized for cleavage by a protease.
[0199] In some embodiments, the multispecific polypeptide construct
contains in order, from N-terminus to C-terminus: an immunoglobulin
Fc region; a linker; a CD3 binding region that binds CD3
(CD3.epsilon.); and an antigen binding domain that binds a
tumor-associated antigen (TAA). In some embodiments, the
multispecific polypeptide construct contains in order, from
N-terminus to C-terminus: an antigen binding domain that binds to a
tumor-associated antigen (TAA); an immunoglobulin Fc region; a
linker; and a CD3 binding region that binds CD3 (CD3.epsilon.). In
some embodiments, the multispecific polypeptide construct contains
at least a first antigen binding domain that binds a TAA and a
second antigen binding domain that binds a TAA. In some
embodiments, the multispecific polypeptide construct contains, in
order, from N-terminus to C-terminus: a first antigen binding
domain that binds to a tumor-associated antigen (TAA); an
immunoglobulin Fc region; a linker; a CD3 binding region that binds
CD3 (CD3.epsilon.); and a second antigen binding domain that binds
a tumor-associated antigen (TAA).
[0200] Each of the components of the multispecific polypeptide
constructs of the disclosure is described in more detail below.
[0201] 1. Anti-CD3 Binding Domains:
[0202] The multispecific polypeptide constructs of the disclosure
include one or more copies of an anti-CD3 binding domain. The
anti-CD3 binding domains of the disclosure activate T cells via
engagement of CD3.epsilon. on the T cells. The anti-CD3 binding
domains of the disclosure agonize, stimulate, activate, and/or
otherwise augment CD3-mediated T cell activation. Biological
activities of CD3 include, for example, T cell activation and other
signaling through interaction between CD3 and the antigen-binding
subunits of the T-Cell Receptor (TCR). For example, the anti-CD3
binding domains of the disclosure completely or partially activate
T cells via engagement of CD3.epsilon. on T cells by partially or
completely modulating, e.g., agonizing, stimulating, activating or
otherwise augmenting CD3-mediated T cell activation.
[0203] In preferred embodiments, the anti-CD3 binding domains of
the disclosure specifically bind the epsilon chain of CD3, also
known as CD3.epsilon.. The anti-CD3.epsilon. binding domains of the
disclosure activate T cells via engagement of CD3.epsilon. on the T
cells. The anti-CD3.epsilon. binding domains of the disclosure
include monoclonal antibodies, such as, for example, mammalian
monoclonal antibodies, primate monoclonal antibodies, fully human
monoclonal antibodies, as well as humanized monoclonal antibodies
and chimeric antibodies, as well as antigen-binding fragments
thereof. In some embodiments, the anti-CD3.epsilon. binding domain
includes one or more copies of an antibody or an antigen-binding
fragment thereof.
[0204] In some embodiments, the anti-CD3.epsilon. binding domain
includes a combination of a VH CDR1 sequence, a VH CDR2 sequence,
and a VH CDR3 sequence, wherein at least one of the VH CDR1
sequence, the VH CDR2 sequence, and the VH CDR3 sequence is
selected from a VH CDR1 sequence that includes at least the amino
acid sequence TYAMN (SEQ ID NO: 16); a VH CD2 sequence that
includes at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ
ID NO: 17); and a VH CDR3 sequence that includes at least the amino
acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18). In some embodiments,
the anti-CD3.epsilon. binding domain includes a combination of a VH
CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein
at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the
VH CDR3 sequence is selected from a VH CDR1 sequence that includes
at least the amino acid sequence GFTFNTYAMN (SEQ ID NO: 211); a VH
CDR2 sequence that includes at least the amino acid sequence
RIRSKYNNYATY (SEQ ID NO: 212); and a VH CDR3 sequence that includes
at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:
18).
[0205] In some embodiments, the anti-CD3.epsilon. binding domain
includes a combination of a VL CDR1 sequence, a VL CDR2 sequence,
and a VL CDR3 sequence, wherein at least one of the VL CDR1
sequence, the VL CDR2 sequence, and the VL CDR3 sequence is
selected from a VL CDR1 sequence that includes at least the amino
acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 sequence
that includes at least the amino acid sequence GTNKRAP (SEQ ID NO:
20); and a VL CDR3 sequence that includes at least the amino acid
sequence ALWYSNLWV (SEQ ID NO: 21).
[0206] In some embodiments, the anti-CD3.epsilon. binding domain
includes a VH CDR1 sequence that includes at least the amino acid
sequence TYAMN (SEQ ID NO: 16); a VH CDR2 sequence that includes at
least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17);
a VH CDR3 sequence that includes at least the amino acid sequence
HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence that includes at
least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL
CDR2 sequence that includes at least the amino acid sequence
GTNKRAP (SEQ ID NO: 20); and a VL CDR3 sequence that includes at
least the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).
[0207] In some embodiments, the anti-CD3.epsilon. binding domain
includes a VH CDR1 sequence that includes at least the amino acid
sequence GFTFNTYAMN (SEQ ID NO: 211); a VH CDR2 sequence that
includes at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:
212); a VH CDR3 sequence that includes at least the amino acid
sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence that
includes at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID
NO: 19); a VL CDR2 sequence that includes at least the amino acid
sequence GTNKRAP (SEQ ID NO: 20); and a VL CDR3 sequence that
includes at least the amino acid sequence ALWYSNLWV (SEQ ID NO:
21).
[0208] In some embodiments, the anti-CD3.epsilon. binding domain
includes a VH CDR1 sequence that includes at least the amino acid
sequence GFTFNTYAMN (SEQ ID NO: 211); a VH CDR2 sequence that
includes at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:
212); a VH CDR3 sequence that includes at least the amino acid
sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence that
includes at least the amino acid sequence GSSTGAVITSNYAN (SEQ ID
NO: 229); a VL CDR2 sequence that includes at least the amino acid
sequence GTNKRAP (SEQ ID NO: 230); and a VL CDR3 sequence that
includes at least the amino acid sequence ALWYSNHWV (SEQ ID NO:
225).
[0209] In some embodiments, the anti-CD3.epsilon. binding domain
includes a combination of a VH CDR1 sequence, a VH CDR2 sequence,
and a VH CDR3 sequence, wherein at least one of the VH CDR1
sequence, the VH CDR2 sequence, and the VH CDR3 sequence is
selected from a VH CDR1 sequence that includes a sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence TYAMN (SEQ ID NO: 16); a VH
CDR2 sequence that includes a sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); and a VH
CDR3 sequence that includes a sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18).
[0210] In some embodiments, the anti-CD3.epsilon. binding domain
includes a combination of a VH CDR1 sequence, a VH CDR2 sequence,
and a VH CDR3 sequence, wherein at least one of the VH CDR1
sequence, the VH CDR2 sequence, and the VH CDR3 sequence is
selected from a VH CDR1 sequence that includes a sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO: 211); a
VH CDR2 sequence that includes a sequence that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to
the amino acid sequence RIRSKYNNYATY (SEQ ID NO: 212); and a VH
CDR3 sequence that includes a sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18).
[0211] In some embodiments, the anti-CD3.epsilon. binding domain
includes a combination of a VL CDR1 sequence, a VL CDR2 sequence,
and a VL CDR3 sequence, wherein at least one of the VL CDR1
sequence, the VL CDR2 sequence, and the VL CDR3 sequence is
selected from a VL CDR1 sequence that includes a sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:
19); a VL CDR2 sequence that includes a sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the amino acid sequence GTNKRAP (SEQ ID NO: 20); and a VL CDR3
sequence that includes a sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino
acid sequence ALWYSNLWV (SEQ ID NO: 21).
[0212] In some embodiments, the anti-CD3.epsilon. binding domain
includes a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence TYAMN (SEQ ID NO: 16); a VH CD2 sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a
VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL
CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP
(SEQ ID NO: 20); and a VL CDR3 sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence ALWYSNLWV (SEQ ID NO: 21).
[0213] In some embodiments, the anti-CD3.epsilon. binding domain
includes a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence GFTFNTYAMN (SEQ ID NO: 211); a VH CDR2 sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO: 212);
a VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL
CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP
(SEQ ID NO: 20); and a VL CDR3 sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence ALWYSNLWV (SEQ ID NO: 21).
[0214] In some embodiments, the anti-CD3.epsilon. binding domain
includes a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence GFTFNTYAMN (SEQ ID NO: 211); a VH CDR2 sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to amino acid sequence RIRSKYNNYATY (SEQ ID NO: 212); a
VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 229); a VL
CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP
(SEQ ID NO: 230); and a VL CDR3 sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence ALWYSNHWV (SEQ ID NO: 225).
[0215] In some embodiments, the anti-CD3.epsilon. binding domain
includes a VH CDR1 sequence that includes at least the amino acid
sequence GFTSTYAMN (SEQ ID NO: 227); a VH CDR2 sequence that
includes at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO:
228); a VH CDR3 sequence that includes at least the amino acid
sequence HGNFGDSYVSWFAY (SEQ ID NO: 224), a VL CDR1 sequence that
includes at least the amino acid sequence GSSTGAVTTSNYAN (SEQ ID
NO: 229); a VL CDR2 sequence that includes at least the amino acid
sequence GTNKRAP (SEQ ID NO: 230); and a VL CDR3 sequence that
includes at least the amino acid sequence ALWYSNHWV (SEQ ID NO:
225).
[0216] In some embodiments, the anti-CD3.epsilon. binding domain
includes a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence GFTFSTYAMN (SEQ ID NO: 227); a VH CDR2 sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence RIRSKYNNYATY (SEQ ID NO: 228);
a VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
HGNFGDSYVTSWFAY (SEQ ID NO: 224), a VL CDR1 sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO:
229); a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence GTNKRAP (SEQ ID NO: 230); and a VL CDR3 sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence ALWYSNHWV (SEQ ID NO:
225).
[0217] In some embodiments, the anti-CD3.epsilon. binding domain
includes a CDR3 that includes at least amino acids VLWYSNRWV (SEQ
ID NO:226). In some embodiments, the anti-CD3.epsilon. binding
domain includes a CDR3 that is at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more identical to the amino acids
VLWYSNRWV (SEQ ID NO:226).
[0218] In some embodiments, the anti-CD3.epsilon. binding domain
includes one or more copies of an antibody or an antigen-binding
fragment thereof selected from the group consisting of a Fab
fragment, a F(ab).sub.2 fragment, an Fv fragment, a scFv, a scAb, a
dAb, a single domain heavy chain antibody, and a single domain
light chain antibody. In some embodiments, the anti-CD3 binding
domain includes an Fv antibody fragment that binds CD3.epsilon.
(referred to herein as an anti-CD3.epsilon. Fv fragment). In some
embodiments, the anti-CD3.epsilon. Fv antibody fragment is a
disulfide stabilized anti-CD3 binding Fv fragment (dsFv). In some
embodiments, the anti-CD3 binding domain is monovalent for binding
CD3.
[0219] In some embodiments, the CD3 binding region is not a single
chain antibody. For example, in some aspects, the CD3 binding
region is not a single chain variable fragment (scFv).
[0220] In some embodiments, the CD3 binding region is an Fv
antibody fragment containing a variable heavy chain (Hv, also
called VH) and variable light chain (Lv, also called VL), such as
any as described. In aspects of such embodiments, the
immunoglobulin Fc region is a heterodimeric Fc region containing
two different Fc polypeptides capable of heterodimeric association
between both polypeptides of the Fc heterodimer, such as any as
described in Section 11.2. In such embodiments, the variable heavy
chain (VH) and variable light chain (VL) of the CD3 binding region
are linked on opposite chains of the heterodimeric Fc.
[0221] In some embodiments, the anti-CD3.epsilon. Fv antibody
fragment includes an amino acid sequence selected from the group of
SEQ ID NO: 32-81. In some embodiments, the anti-CD3.epsilon. Fv
antibody fragment includes an amino acid sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to an amino acid sequence selected from the group consisting of SEQ
ID NO: 32-81. In some embodiments, the anti-CD3.epsilon. Fv
antibody fragment includes a combination of an amino acid sequence
selected from the group of SEQ ID NO: 32-62 and an amino acid
sequence selected from the group consisting of SEQ ID NO: 63-81. In
some embodiments, the anti-CD3.epsilon. Fv antibody fragment
includes a combination of an amino acid sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to an amino acid sequence selected from the group consisting of SEQ
ID NO: 32-62 and an amino acid sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an
amino acid sequence selected from the group consisting of SEQ ID
NO: 63-81 an amino acid sequence.
[0222] In some embodiments, the anti-CD3.epsilon. binding domain
thereof includes a combination of a heavy chain variable region
amino acid sequence and a light chain variable region amino acid
sequence comprising an amino acid sequence selected from the group
of SEQ ID NO: 32-81. In some embodiments, the anti-CD3.epsilon.
binding domain thereof includes a combination of a heavy chain
variable region amino acid sequence selected from the group of SEQ
ID NO: 32-62 and a light chain variable region amino acid sequence
comprising an amino acid sequence selected from the group of SEQ ID
NO: 63-81.
[0223] In some embodiments, the anti-CD3.epsilon. binding domain
thereof is an Fv fragment that includes a combination of heavy
chain variable amino acid sequence and a light chain variable amino
acid sequence. In some embodiments, the anti-CD3.epsilon. binding
domain thereof is an Fv fragment that includes a combination of
heavy chain variable amino acid sequence and a light chain variable
amino acid sequence comprising an amino acid sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 14, 15, 32-81, 191, 196-200, 211, and
212. In some embodiments, the anti-CD3.epsilon. binding domain
thereof is an Fv fragment that includes a combination of heavy
chain variable amino acid sequence and a light chain variable amino
acid sequence comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 14, 15, 32-81, 191, 196-200, 211,
and 212. In some embodiments, the anti-CD3.epsilon. binding domain
thereof is an Fv fragment that includes a combination of heavy
chain variable amino acid sequence selected from the group of SEQ
ID NO: 14, 32-62, 196-198, and 211 and light chain variable amino
acid sequence selected from the group consisting of SEQ ID NO: 15,
63-81, 191, 199, 200, and 212. In some embodiments, the
anti-CD3.epsilon. binding domain thereof is an Fv fragment that
includes a combination of heavy chain variable amino acid sequence
that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or more identical to an amino acid sequence selected from the group
consisting of SEQ ID NO: 14, 32-62, 196-198, and 211 and a light
chain variable amino acid sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino
acid sequence selected from the group consisting of SEQ ID NO: 15,
63-81, 191, 199, 200, and 212.
[0224] In some embodiments, the anti-CD3.epsilon. binding domain
thereof includes a combination of a heavy chain variable region
amino acid sequence and a light chain variable region amino acid
sequence comprising an amino acid sequence selected from the group
of SEQ ID NO: 32-81, 191, 196-200, 211, and 212. In some
embodiments, the anti-CD3.epsilon. binding domain thereof includes
a combination of a heavy chain variable region amino acid sequence
selected from the group of SEQ ID NO: 32-62, 196-198, and 211 and a
light chain variable region amino acid sequence comprising an amino
acid sequence selected from the group of SEQ ID NO: 63-81, 191,
199, 200, and 212.
[0225] In some embodiments, the anti-CD3.epsilon. Fv antibody
fragment includes a combination of an amino acid sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to an amino acid sequence selected from the group
consisting of SEQ ID NO: 14, 32-43, 45-47, 48, 196 and 211 and an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to an amino acid sequence
selected from the group consisting of SEQ ID NO: 15, 63, 65-71, 73,
75, 77, and 199. In some embodiments, the anti-CD3.epsilon. Fv
antibody fragment includes a combination of an amino acid sequence
selected from the group of SEQ ID NO: 14, 32-43, 45-47, 48, 196 and
211 and an amino acid sequence selected from the group consisting
of SEQ ID NO: 15, 63, 65-71, 73, 75, 77, and 199.
[0226] In some embodiments, the anti-CD3.epsilon. binding domain
thereof includes a variable heavy chain (VH) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 14. In some embodiments, the anti-CD3.epsilon. binding
domain includes a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 15. In some embodiments, the anti-CD3.epsilon. binding
domain thereof includes a variable heavy chain (VH) comprising an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence of
SEQ ID NO: 14 and a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 15. In some embodiments, the anti-CD3.epsilon. binding
domain thereof includes a variable heavy chain (VH) comprising the
amino acid sequence of SEQ ID NO: 14. In some embodiments, the
anti-CD3.epsilon. binding domain includes a variable light chain
(VL) comprising the amino acid sequence of SEQ ID NO: 15. In some
embodiments, the anti-CD3.epsilon. binding domain thereof includes
a variable heavy chain (VH) comprising the amino acid sequence of
SEQ ID NO: 14 and a variable light chain (VL) comprising the amino
acid sequence of SEQ ID NO: 15.
[0227] In some embodiments, the anti-CD3.epsilon. binding domain
thereof includes a variable heavy chain (VH) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 196. In some embodiments, the anti-CD3.epsilon. binding
domain includes a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 199. In some embodiments, the anti-CD3.epsilon. binding
domain thereof includes a variable heavy chain (VH) comprising an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence of
SEQ ID NO: 196 and a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 199. In some embodiments, the anti-CD3.epsilon. binding
domain thereof includes a variable heavy chain (VH) comprising the
amino acid sequence of SEQ ID NO: 196. In some embodiments, the
anti-CD3.epsilon. binding domain includes a variable light chain
(VL) comprising the amino acid sequence of SEQ ID NO: 199. In some
embodiments, the anti-CD3.epsilon. binding domain thereof includes
a variable heavy chain (VH) comprising the amino acid sequence of
SEQ ID NO: 196 and a variable light chain (VL) comprising the amino
acid sequence of SEQ ID NO: 199.
[0228] In particular embodiments, the Fv is a disulfide stabilized
Fv fragment (dsFv) in which the V.sub.H-V.sub.L heterodimer is
stabilized by an interchain disulfide bond. In some embodiments,
the interchain disulfide bond is engineered by mutation of position
in framework positions of the VH and/or VL chain. In some
embodiments, the disulfide stabilized anti-CD3 Fv comprises an
anti-CD3 VH with the mutation 44 to Cys and an anti-CD3 VL with the
mutation 100 to Cys by Kabat numbering. For example, in some
embodiments, the VH chain contains the mutation G44C and the VL
chain contains the mutation G100C, each by kabat numbering. In some
embodiments, the disulfide stabilized anti-CD3 Fv comprises an
anti-CD3 VH with the mutation at position 105 to Cys and an
anti-CD3 VL with the mutation position 43 to Cys by Kabat
numbering.
[0229] In some embodiments, the anti-CD3.epsilon. Fv antibody
fragment includes a combination of an amino acid sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to an amino acid sequence selected from the group
consisting of SEQ ID NO: 44, 49-62, 197 and 198 and an amino acid
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to an amino acid sequence selected from
the group consisting of SEQ ID NO: 64, 72, 74, 76, 78-81, 191, 200
and 212. In some of any such embodiments, the anti-CD3 Fv is a dsFv
that has a VH chain containing the mutation G44C and a VL chain
containing the mutation G100C, each by kabat numbering. In some
embodiments, the anti-CD3.epsilon. Fv antibody fragment includes a
combination of an amino acid sequence selected from the group of
SEQ ID NO: 44, 49-62, 197 and 198 and an amino acid sequence
selected from the group consisting of SEQ ID NO: 64, 72, 74, 76,
78-81, 191, 200 and 212.
[0230] In some embodiments, the anti-CD3.epsilon. binding domain
thereof includes a variable heavy chain (VH) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 44. In some embodiments, the anti-CD3.epsilon. binding
domain includes a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 72. In some embodiments, the anti-CD3.epsilon. binding
domain thereof includes a variable heavy chain (VH) comprising an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence of
SEQ ID NO: 44 and a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 72. In some of any such embodiments, the anti-CD3 Fv is a
dsFv that has a VH chain containing the mutation G44C and a VL
chain containing the mutation G100C, each by kabat numbering. In
some embodiments, the anti-CD3.epsilon. binding domain thereof
includes a variable heavy chain (VH) comprising the amino acid
sequence of SEQ ID NO: 44. In some embodiments, the
anti-CD3.epsilon. binding domain includes a variable light chain
(VL) comprising the amino acid sequence of SEQ ID NO: 72. In some
embodiments, the anti-CD3.epsilon. binding domain thereof includes
a variable heavy chain (VH) comprising the amino acid sequence of
SEQ ID NO: 44 and a variable light chain (VL) comprising the amino
acid sequence of SEQ ID NO: 72.
[0231] In some embodiments, the anti-CD3.epsilon. binding domain
thereof includes a variable heavy chain (VH) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 198. In some embodiments, the anti-CD3.epsilon. binding
domain includes a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 200. In some embodiments, the anti-CD3.epsilon. binding
domain thereof includes a variable heavy chain (VH) comprising an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence of
SEQ ID NO: 198 and a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 200. In some of any such embodiments, the anti-CD3 Fv is a
dsFv that has a VH chain containing the mutation G44C and a VL
chain containing the mutation G100C, each by kabat numbering. In
some embodiments, the anti-CD3.epsilon. binding domain thereof
includes a variable heavy chain (VH) comprising the amino acid
sequence of SEQ ID NO: 198. In some embodiments, the
anti-CD3.epsilon. binding domain includes a variable light chain
(VL) comprising the amino acid sequence of SEQ ID NO: 200. In some
embodiments, the anti-CD3.epsilon. binding domain thereof includes
a variable heavy chain (VH) comprising the amino acid sequence of
SEQ ID NO: 198 and a variable light chain (VL) comprising the amino
acid sequence of SEQ ID NO: 200.
[0232] In some embodiments, the anti-CD3.epsilon. binding domain
thereof includes a variable heavy chain (VH) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 197. In some embodiments, the anti-CD3.epsilon. binding
domain includes a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 200. In some embodiments, the anti-CD3.epsilon. binding
domain thereof includes a variable heavy chain (VH) comprising an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence of
SEQ ID NO: 197 and a variable light chain (VL) comprising an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 200. In some of any such embodiments, the anti-CD3 Fv is a
dsFv that has a VH chain containing the mutation G44C and a VL
chain containing the mutation G100C, each by kabat numbering. In
some embodiments, the anti-CD3.epsilon. binding domain thereof
includes a variable heavy chain (VH) comprising the amino acid
sequence of SEQ ID NO: 197. In some embodiments, the
anti-CD3.epsilon. binding domain includes a variable light chain
(VL) comprising the amino acid sequence of SEQ ID NO: 200. In some
embodiments, the anti-CD3.epsilon. binding domain thereof includes
a variable heavy chain (VH) comprising the amino acid sequence of
SEQ ID NO: 197 and a variable light chain (VL) comprising the amino
acid sequence of SEQ ID NO: 200.
[0233] 2. Immunoglobulin Fc Polypeptides:
[0234] The first component of the multispecific polypeptide
constructs of the disclosure includes an immunoglobulin Fc region.
In some embodiments, the immunoglobulin Fc region is an IgG isotype
selected from the group consisting of IgG1 isotype, IgG2 isotype,
IgG3 isotype, and IgG4 subclass. In some embodiments, the Fc region
is a human Fc. In some embodiments, the immunoglobulin Fc region is
a polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 1-6. In some embodiments, the
immunoglobulin Fc region contains an Fc chain that is an
immunologically active fragment of any of SEQ ID Nos: 1-6. In some
embodiments, the immunoglobulin Fc region contains an Fc
polypeptide chain that is at least 50%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to the amino acid sequence of any of SEQ ID NOs: 1-6 or an
immunologically active fragment thereof.
[0235] In some embodiments, the multispecific polypeptide construct
is a dimer formed by polypeptides, each containing an Fc. In some
specific embodiments, identical or substantially identical
polypeptides will be dimerized to create a homodimer. In some
embodiments, the dimer is a homodimer in which the two polypeptides
of the multispecific polypeptide construct are the same. In other
cases, the Fc region is formed by Fc domains that are mutated or
modified to promote heterodimerization in which different
polypeptides can be dimerized to yield a heterodimer. Thus, in some
embodiments, the dimer is a heterodimer in which two polypeptide
chains of the multispecific polypeptide construct are different.
Exemplary modifications to promote heterodimerization are known,
including any as described below.
[0236] In general, the Fc region is responsible for effector
functions, such as complement-dependent cytotoxicity (CDC) and
antibody-dependent cell cytotoxicity (ADCC), in addition to the
antigen-binding capacity, which is the main function of
immunoglobulins. Additionally, the FcRn sequence present in the Fc
region plays the role of regulating the IgG level in serum by
increasing the in vivo half-life by conjugation to an in vivo FcRn
receptor. In some embodiments, such functions can be altered, such
as reduced or enhanced, in an Fc for use with the provided
multispecific polypeptide constructs.
[0237] In some embodiments, the Fc region of the provided
multispecific polypeptide constructs exhibit one or more effector
functions. In some cases, the Fc region is capable of providing
Fc-mediated effector functions, such as for example, ADCC (e.g.,
release of granzyme B by NK cells), ADCP, and/or CDC. Thus, in some
embodiments in which the multispecific polypeptide constructs
contain a cleavable linker, cleavage of the linker can produce two
components that each have biological activity: the CD3-binding
region that is able to bind and engage CD3 on a T cell and the Fc
region linked to the TAA-antigen binding domain that can exhibit
target-specific effector function. In particular embodiments
provided herein, the multispecific polypeptide constructs contain a
non-cleavable linker and may, in some aspects, not exhibit an
independent Fc-mediated effector function.
[0238] In some embodiments, the Fc region includes an Fc
polypeptide that is mutated or modified to alter one or more
effector functions. Various examples of mutations to Fc
polypeptides to alter, such as reduce, effector function are known,
including any as described below. In some embodiments, reference to
amino acid substitutions in an Fc region is by EU numbering by
Kabat (also called Kabat numbering) unless described with reference
to a specific SEQ ID NO. EU numbering is known and is according to
the most recently updated IMGT Scientific Chart (IMGT.RTM., the
international ImMunoGeneTics information System.RTM.,
http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html
(created: 17 May 2001, last updated: 10 Jan. 2013) and the EU index
as reported in Kabat, E. A. et al. Sequences of Proteins of
Immunological interest. 5th ed. US Department of Health and Human
Services, NIH publication No. 91-3242 (1991).
[0239] In some embodiments, provided multispecific polypeptide
constructs that contain an Fc region that exhibits reduced effector
functions, may be a desirable candidate for applications in which
constrained CD3 binding is desired yet certain effector functions
(such as CDC and ADCC) are unnecessary or deleterious. In vitro
and/or in vivo cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
multispecific polypeptide constructs and/or cleaved components
thereof lack Fc.gamma.R binding (hence likely lacking ADCC
activity), but retains FcRn binding ability. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII.
Non-limiting examples of in vitro assays to assess ADCC activity of
a molecule of interest is described in U.S. Pat. No. 5,500,362
(see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA
83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad.
Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see
Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-radioactive assay methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.TM. non-radioactive cytotoxicity assay (Promega, Madison, Wis.).
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). C1q binding assays may also be carried out to
confirm that the multispecific polypeptide construct or cleaved
components thereof is unable to bind C1q and hence lacks CDC
activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood
101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood
103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769
(2006)).
[0240] In some embodiments, the immunoglobulin Fc region or
immunologically active fragment thereof is an IgG isotype. For
example, the immunoglobulin Fc region of the fusion protein is of
human IgG1 isotype, having an amino acid sequence:
TABLE-US-00001 (SEQ ID NO: 1) ##STR00001## ##STR00002## APIEKTISKA
KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD
SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK
[0241] In some embodiments, the immunoglobulin Fc region or
immunologically active fragment thereof comprises a human IgG1
polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to the amino acid sequence of SEQ ID NO: 1.
[0242] In some embodiments, an IgG1 Fc polypeptide or a variant
thereof such as any described below can be made in a G1 ml or G1 m3
allotype. In some embodiments, the Fc region can contain amino
acids of the human G1 ml allotype, such as residues containing Asp
(D) and Leu (L) at positions 356 and 358, e.g. as set forth in SEQ
ID NO:1. In some cases, an Fc polypeptide can contain amino acid
substitutions E356D and M358L to reconstitute residues of allotype
G1 ml. In other embodiments, the Fc region can contain amino acids
of the human G1 m3 allotype, such as residues Glu (E) and Met (M)
at positions 356 and 358 by EU numbering, e.g. as set forth in SEQ
ID NOS: 194 and 195. In some cases, an Fc polypeptide can contain
amino acid substitutions D356E and L358M to reconstitute residues
of allotype G1 m3. In some embodiments, the human IgG1 Fc region is
modified to alter antibody-dependent cellular cytotoxicity (ADCC)
and/or complement-dependent cytotoxicity (CDC), e.g., the amino
acid modifications described in Natsume et al., 2008 Cancer Res,
68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5;
Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS,
103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604;
Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen
et al., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al, 1992
J Immunol, 148: 3461-3468; Reviewed in Kaneko and Niwa, 2011
Biodrugs, 25(1):1-11, the contents of each of which are hereby
incorporated by reference in their entireties.
[0243] In some embodiments, the Fc region, such as the human IgG1
Fc region is modified to enhance ADCC activity or CDC activity.
Examples of mutations that enhance ADCC include modification at
Ser239 and Ile332, for example Ser239Asp and Ile332Glu (S239D,
1332E). Examples of mutations that enhance CDC include
modifications at Lys326 and Glu333. In some embodiments, the Fc
region is modified at one or both of these positions, for example
Lys326Ala and/or Glu333Ala (K326A and E333A) using the Kabat
numbering system.
[0244] In some embodiments, the human IgG1 Fc region fusion
proteins of the present disclosure lack or have reduced Fucose
attached to the N-linked glycan-chain at N297. There are numerous
ways to prevent fucosylation, including but not limited to
production in a FUT8 deficient cell line; addition inhibitors to
the mammalian cell culture media, for example Castanospermine; and
metabolic engineering of the production cell line. In some
embodiments, the human IgG1 Fc region is modified at amino acid
Asn297 (Boxed, Kabat Numbering) to prevent glycosylation of the
fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D).
[0245] In some embodiments, the Fc region is altered to provide
reduced Fc-mediated effector functions, such as via reduced Fc
receptor binding, e.g. binding to Fc.gamma.R binding but generally
not FcRn binding. In some embodiments, the Fc region of the fusion
protein is altered at one or more of the following positions to
reduce Fc receptor binding: Leu 234 (L234), Leu235 (L235), Asp265
(D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325)
orAla327 (A327). For example, Leu 234Ala (L234A), Leu235Ala
(L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N),
Asn297Ala (N297A), Asn325Glu (N325E) orAla327Ser (A327S). In some
embodiments, the Fc region of the fusion protein is modified at
amino acid Leu235 (Boxed in SEQ ID NO:1 above, Kabat Numbering) to
alter Fc receptor interactions, e.g., Leu235Glu (L235E) or
Leu235Ala (L235A). In some embodiments, the Fc region of the fusion
protein is modified at amino acid Leu234 (Boxed in SEQ ID NO:1
above, Kabat Numbering) to alter Fc receptor interactions, e.g.,
Leu234Ala (L234A). In some embodiments, the Fc region of the fusion
protein is altered at both amino acid 234 and 235, e.g., Leu234Ala
and Leu235Ala (L234A/L235A) or Leu234Val and Leu235Ala
(L234V/L235A). In preferred embodiments, modifications within the
Fc region reduce binding to Fc-receptor-gamma receptors while have
minimal impact on binding to the neonatal Fc receptor (FcRn).
[0246] In some embodiments, the human IgG Fc region is modified to
enhance FcRn binding. Examples of Fc mutations that enhance binding
to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E,
respectively) (Kabat numbering, Dall'Acqua et al 2006, 1 Biol Chem
Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S)
(Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159) (EU index
of Kabat et al 1991 Sequences of Proteins of Immunological
Interest). In some embodiments, the mutated or modified Fc
polypeptide includes the following mutations: Met252Tyr and
Met428Leu or Met252Tyr and Met428Val (M252Y, M428L, or M252Y,
M428V) using the Kabat numbering system.
[0247] In some embodiments, the Fc region of the fusion protein is
lacking an amino acid at one or more of the following positions to
reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235
(L235). In these embodiments, Fc deletion of these three amino
acids reduces the complement protein C1q binding.
TABLE-US-00002 (SEQ ID NO: 2) PAPGGPSVFL FPPKPKDTLM ISRTPEVTCV
VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE
SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS
LSPGK
[0248] In some embodiments, the Fc region is mutated in one or more
of the following positions to reduce Fc receptor binding: Glu233
(E233), Leu234 (L234), or Leu235 (L235). The one or more mutations
can include E233P, L234V and/or L235A.
[0249] In some embodiments, the Fc region of the fusion protein is
altered at Gly236 (boxed in SEQ ID NO:1 above) to reduce Fc
receptor binding. For example, wherein Gly236 is deleted from the
fusion protein. In some embodiments, the human IgG1 Fc region is
modified at amino acid Gly236 to enhance the interaction with
CD32A, e.g., Gly236Ala (G236A).
[0250] In particular embodiments, the mutations of the Fc region to
reduce Fc effector function, e.g. via reducing Fc receptor binding
to Fc.gamma.R, include mutations from among any of G236R/L328R,
E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,
E233P/L234V/L235A/G236del/S239K/A327G,
E233P/L234V/L235A/G236del/S267K/A327G or
E233P/L234V/L235A/G236del.
[0251] In some embodiments, the human IgG1 Fc region lacks Lys447
(EU index of Kabat et al 1991 Sequences of Proteins of
Immunological Interest).
[0252] In some embodiments, the fusion or immunologically active
fragment thereof comprises a human IgG2 polypeptide sequence that
is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence of SEQ ID NO: 2.
[0253] In some embodiments, the immunoglobulin Fc region or
immunologically active fragment of the fusion protein is of human
IgG2 isotype, having an amino acid sequence:
TABLE-US-00003 (SEQ ID NO: 3) PAPPVAGPSV FLFPPKPKDT LMISRTPEVT
CVVVDVSHED PEVQFNWYVD ##STR00003## PIEKTISKTK GQPREPQVYT LPPSREEMTK
NQVSLTCLVK GFYPSDISVE WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG
NVFSCSVMHE ALHNHYTQKS LSLSPGK
[0254] In some embodiments, the fusion or immunologically active
fragment thereof comprises a human IgG2 polypeptide sequence that
is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence of SEQ ID NO: 3.
[0255] In some embodiments, the human IgG2 Fc region is modified at
amino acid Asn297 (Boxed, to prevent to glycosylation of the
antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some
embodiments, the human IgG2 Fc region lacks Lys447 (EU index of
Kabat et al 1991 Sequences of Proteins of Immunological
Interest).
[0256] In some embodiments, the immunoglobulin Fc region or
immunologically active fragment of the fusion protein is of human
IgG3 isotype, having an amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 4) PAPELLGGPS VFLFPPKPKD TLMISRTPEV
TCVVVDVSHE DPEVQFKWYV ##STR00004## APIEKTISKT KGQPREPQVY TLPPSREEMT
KNQVSLTCLV KGFYPSDIAV EWESSGQPEN NYNTTPPMLD SDGSFFLYSK LTVDKSRWQQ
GNIFSCSVMH ##STR00005##
[0257] In some embodiments, the antibody or immunologically active
fragment thereof comprises a human IgG3 polypeptide sequence that
is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence of SEQ ID NO: 4.
[0258] In some embodiments, the human IgG3 Fc region is modified at
amino acid Asn297 (Boxed, Kabat Numbering) to prevent to
glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp
(N297D). In some embodiments, the human IgG3 Fc region is modified
at amino acid 435 to extend the half-life, e.g., Arg435His (R435H).
In some embodiments, the human IgG3 Fc region lacks Lys447 (EU
index of Kabat et al 1991 Sequences of Proteins of Immunological
Interest).
[0259] In some embodiments, the immunoglobulin Fc region or
immunologically active fragment of the fusion protein is of human
IgG4 isotype, having an amino acid sequence:
TABLE-US-00005 (SEQ ID NO: 5) ##STR00006## ##STR00007## SSIEKTISKA
KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD
SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK
[0260] In some embodiments, the antibody or immunologically active
fragment thereof comprises a human IgG4 polypeptide sequence that
is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence of SEQ ID NO: 5.
[0261] In some embodiments, the immunoglobulin Fc region or
immunologically active fragment of the fusion protein is of human
IgG4 isotype, having an amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 6) PAPELLGGPS VFLFPPKPKD TLMISRTPEV
TCVVVDVSQE DPEVQFNWYV ##STR00008## SSIEKTISKA KGQPREPQVY TLPPSQEEMT
KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE
GNVFSCSVMH EALHNHYTQK SLSLSLGK
[0262] In some embodiments, the antibody or immunologically active
fragment thereof comprises a human IgG4 polypeptide sequence that
is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence of SEQ ID NO: 6.
[0263] In other embodiments, the human IgG4 Fc region is modified
at amino acid 235 to alter Fc receptor interactions, e.g.,
Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region is
modified at amino acid Asn297 (Boxed, Kabat Numbering) to prevent
to glycosylation of the antibody, e.g., Asn297Ala (N297A) or
Asn297Asp (N297D). In some embodiments, the human IgG4 Fc region
lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of
Immunological Interest).
[0264] In some embodiments, the human IgG Fc region is modified to
stabilize the homodimerization at the CH3:CH3 interface by
introducing two disulfide bonds by changing Ser354 to Cys (S354C)
and Tyr349 to Cys (Y349C) (S354C/Y349C).
[0265] In particular embodiments of multispecific polypeptide
constructs provided herein, the human IgG Fc region is modified to
induce heterodimerization. Various methods are known for promoting
heterodimerization of complementary Fc polypeptides, see e.g.
Ridgway et al, Protein Eng. 9:617-621 (1996); Merchant et al, Nat.
Biotechnol. 16(7): 677-81 (1998); Moore et al. (2011) MAbs,
3:546-57; Von Kreudenstein et al. MAbs, (2013) 5:646-54;
Gunasekaran et al. (2010) J. Biol. Chem., 285:19637-46; Leaver-Fay
et al. (2016) Structure, 24:641-51; Ha et al. (2016) Frontiers in
Immunology, 7:1; Davis et al. (2010) Protein Eng Des Sel,
23:195-202; published international PCT Appl. No. WO 1998/050431,
WO 2009/089004, WO2011143545 WO 2014/067011, WO 2012/058768,
WO2018027025; published U.S. patent Appl. No. US20140363426,
US20150307628, US20180016354, US20150239991; and U.S. patent Nos.
U.S. Pat. Nos. 5,731,168, 7,183,076, 9,701,759, 9,605,084, and
9,650,446. Methods to promote heterodimerization of Fc chains
include mutagenesis of the Fc region, such as by including a set of
"knob-into-hole" mutations or including mutations to effect
electrostatic steering of the Fc to favor attractive interactions
among different polypeptide chains. For example, in some
embodiments, the Fc polypeptides of a heterodimer includes a
mutation to alter charge polarity across the Fc dimer interface
such that coexpression of electrostatically matched Fc chains
support favorable attractive interactions thereby promoting desired
Fc heterodimer formation, whereas unfavorable repulsive charge
interactions suppress unwanted Fc homodimer formation (Guneskaran
et al. (2010) JBC, 285: 19637-19646). When co-expressed in a cell,
association between the chains is possible but the chains do not
substantially self-associate due to charge repulsion. Other
strategies for generating a heterodimeric Fc include mixing human
IgG and IgA CH3 domain segments to create a complementary CH3
heterodimer, which is referred to as a SEED Fc.
[0266] Methods and variants for heterodimerization also include
those described in published international PCT App. WO2014/145806,
including "knobs and holes" mutations (also called "skew"
variants), mutations that relate to "electrostatic steering" or
"charge pairs," and pI variants. Heterodimeric variants also
include any as described in U.S. published Appl. No. US2012/0149876
or US2018/011883.
[0267] In some embodiments, to promote heterodimerization both
polypeptides of the Fc heterodimer contain paired or complementary
amino acid modifications. Exemplary paired amino acid modification
of polypeptides of an Fc fusion are set forth in Table 1.
TABLE-US-00007 TABLE 1 Paired amino acids of Heterodimeric Fc First
Fc polypeptide Second Fc Polypeptide T366W T366S/L368W/Y407V
T366W/S354C T366S/L368A/Y407V/Y349C S364H/F405A Y349T/Y349F
T350V/L351Y/F405A/Y407V T350V/T366L/K392L/T394W K360D/D399M/Y407A
E345R/Q347R/T366V/K409V K409D/K392D D399K/E356K K360E/K409W
Q347R/D399V/F405T L360E/K409W/Y349C Q347R/399V/F405T/S354C
K370E/K409W E357N/D399V/F405T
[0268] In some embodiments, modifications include introduction of a
protuberance (knob) into a first Fc polypeptide and a cavity (hole)
into a second Fc polypeptide such that the protuberance is
positionable in the cavity to promote complexing of the first and
second Fc-containing polypeptides. Amino acids targeted for
replacement and/or modification to create protuberances or cavities
in a polypeptide are typically interface amino acids that interact
or contact with one or more amino acids in the interface of a
second polypeptide.
[0269] In some embodiments, a first Fc polypeptide that is modified
to contain protuberance (hole) amino acids include replacement of a
native or original amino acid with an amino acid that has at least
one side chain which projects from the interface of the first Fc
polypeptide and is therefore positionable in a compensatory cavity
(hole) in an adjacent interface of a second polypeptide. Most
often, the replacement amino acid is one which has a larger side
chain volume than the original amino acid residue. One of skill in
the art knows how to determine and/or assess the properties of
amino acid residues to identify those that are ideal replacement
amino acids to create a protuberance. In some embodiments, the
replacement residues for the formation of a protuberance are
naturally occurring amino acid residues and include, for example,
arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W).
In some examples, the original residue identified for replacement
is an amino acid residue that has a small side chain such as, for
example, alanine, asparagine, aspartic acid, glycine, serine,
threonine, or valine.
[0270] In some embodiments, a second Fc polypeptide that is
modified to contain a cavity (hole) is one that includes
replacement of a native or original amino acid with an amino acid
that has at least one side chain that is recessed from the
interface of the second polypeptide and thus is able to accommodate
a corresponding protuberance from the interface of a first
polypeptide. Most often, the replacement amino acid is one which
has a smaller side chain volume than the original amino acid
residue. One of skill in the art knows how to determine and/or
assess the properties of amino acid residues to identify those that
are ideal replacement residues for the formation of a cavity.
Generally, the replacement residues for the formation of a cavity
are naturally occurring amino acids and include, for example,
alanine (A), serine (S), threonine (T) and valine (V). In some
examples, the original amino acid identified for replacement is an
amino acid that has a large side chain such as, for example,
tyrosine, arginine, phenylalanine, or tryptophan.
[0271] The CH3 interface of human IgG1, for example, involves
sixteen residues on each domain located on four anti-parallel
.beta.-strands which buries 1090 .ANG.2 from each surface (see
e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller
et al., (1990) J Mol. Biol., 216, 965-973; Ridgway et al., (1996)
Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168). Modifications
of a CH3 domain to create protuberances or cavities are described,
for example, in U.S. Pat. No. 5,731,168; International Patent
Applications WO98/50431 and WO 2005/063816; and Ridgway et al.,
(1996) Prot. Engin., 9: 617-621. In some examples, modifications of
a CH3 domain to create protuberances or cavities are typically
targeted to residues located on the two central anti-parallel
.beta.-strands. The aim is to minimize the risk that the
protuberances which are created can be accommodated by protruding
into the surrounding solvent rather than being accommodated by a
compensatory cavity in the partner CH3 domain.
[0272] For example, in some embodiments the heterodimeric Fc
includes a polypeptide having an amino acid modification within the
CH3 domain at Thr366, which when replaced with a more bulky amino
acid, e.g., Try (T366W), is able to preferentially pair with a
second CH3 domain having amino acid modifications to less bulky
amino acids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala
and Val, respectively (T366S/L368A/Y407V). Heterodimerization via
CH3 modifications can be further stabilized by the introduction of
a disulfide bond, for example by changing Ser354 to Cys (S354C) and
Tyr349 to Cys (Y349C) on opposite CH3 domains (Reviewed in Carter,
2001 Journal of Immunological Methods, 248: 7-15).
[0273] In particular embodiments, a multispecific polypeptide
construct contains a first and second Fc able to mediate Fc
heterodimerization contains a first Fc polypeptide containing
mutations T366W and S354C and a second Fc polypeptide containing
mutations T366S, L368A, Y407V and Y349C. In some embodiments, the
first Fc polypeptide is selected from an Fc polypeptide comprising
the sequence set forth in SEQ ID NO: 201 or 207 and the second Fc
polypeptide is selected from an Fc polypeptide comprising the
sequence set forth in SEQ ID NO: 202, 205 or 209. In some
embodiments, the first Fc polypeptide is or comprises the sequence
of amino acids set forth in any of SEQ ID NOS: 82, 86, 94 or 96 and
the second Fc polypeptide is or comprises the sequence of amino
acids set forth in any of SEQ ID NOS: 83, 87, 90, 92, 98 or
100.
[0274] In some embodiments, the Fc polypeptide exhibits features
providing Fc-mediated effector functions. In particular examples,
the first Fc polypeptide is or comprises the sequence set forth in
SEQ ID NOs:201 and a second Fc polypeptide that is or comprises SEQ
ID NO: 202 or 205. In some embodiments, the first Fc polypeptide is
or comprises the sequence set forth in SEQ ID NO: 82 and the second
Fc polypeptide is or comprises the sequence set forth in SEQ ID NO:
83 or 90. In some embodiments, the first Fc polypeptide is or
comprises the sequence set forth in SEQ ID NO: 86 and the second Fc
polypeptide is or comprises the sequence set forth in SEQ ID NO: 87
or 92. The first and second Fc polypeptide can be formatted on
either polypeptide chain of the construct.
[0275] In some embodiments, one or both of the first and second Fc
polypeptides can further include one or more amino acid mutations
to further reduce one or more Fc effector functions, such as
reduced Fc receptor binding. Exemplary mutations to reduce Fc
effector functions include any as described. In some embodiments,
the modification can be a deletion of one or more positions Glu233
(E233), Leu234 (L234), or Leu235 (L235), such as a deletion of
amino acids Glu233 (E233), Leu234 (L234), and Leu235 (L235). In
some embodiments, the first Fc polypeptide is selected from an Fc
polypeptide comprising the sequence set forth in SEQ ID NO: 203 or
208 and the second Fc polypeptide is selected from an Fc
polypeptide comprising the sequence set forth in SEQ ID NO: 204,
206 or 210. In some embodiments, the first Fc polypeptide is or
comprises the sequence of amino acids set forth in any of SEQ ID
NOS: 84, 88, 95 or 97 and the second Fc polypeptide is or comprises
the sequence of amino acids set forth in any of SEQ ID NOS: 85, 89,
91, 93, 99 or 101.
[0276] In particular examples, the first Fc polypeptide is or
comprises the sequence set forth in SEQ ID NOs:203 and a second Fc
polypeptide that is or comprises SEQ ID NO: 204 or 206. In some
embodiments, the first Fc polypeptide is or comprises the sequence
set forth in SEQ ID NO: 84 and the second Fc polypeptide is or
comprises the sequence set forth in SEQ ID NO: 85 or 91. In some
embodiments, the first Fc polypeptide is or comprises the sequence
set forth in SEQ ID NO: 88 and the second Fc polypeptide is or
comprises the sequence set forth in SEQ ID NO: 89 or 93. The first
and second Fc polypeptide can be formatted on either polypeptide
chain of the construct.
[0277] In some embodiments, the first Fc polypeptide or second Fc
polypeptide further includes mutations M252Y and/or M428V. In
particular examples, the first Fc polypeptide is or comprises the
sequence set forth in SEQ ID NO:207 and the second Fc polypeptide
is or comprises the sequence set forth in SEQ ID NO:209. In some
embodiments, the first Fc polypeptide is or comprises the sequence
set forth in SEQ ID NO:94 and the second Fc polypeptide is or
comprises the sequence set forth in SEQ ID NO: 98. In some
embodiments, the first Fc polypeptide is or comprises the sequence
set forth in SEQ ID NO:96 and the second Fc polypeptide is or
comprises the sequence set forth in SEQ ID NO: 100. In other
examples, the first Fc polypeptide is or comprises the sequence set
forth in SEQ ID NO:208 and the second Fc polypeptide is or
comprises the sequence set forth in SEQ ID NO:210. In some
embodiments, the first Fc polypeptide is or comprises the sequence
set forth in SEQ ID NO:95 and the second Fc polypeptide is or
comprises the sequence set forth in SEQ ID NO: 99. In some
embodiments, the first Fc polypeptide is or comprises the sequence
set forth in SEQ ID NO:97 and the second Fc polypeptide is or
comprises the sequence set forth in SEQ ID NO: 101. The first and
second Fc polypeptide can be formatted on either polypeptide chain
of the construct.
[0278] Additional examples of variants that can facilitate the
promotion of heterodimers are any combination or pair of steric
variants (e.g. skew variants) of a first Fc polypeptide and a
second Fc polypeptide from among: S364K/E357Q and L368D/K370S;
L368D/K370S and S364K; L368E/K370S and S364K; T411T/E360E/Q362E and
D401K; L368D/K370S and S364K/E357L, K370S and S364K/E357Q and
T366S/L368A/Y407V and T366W or 366S/L368A/Y407V/Y349C and
T366W/S354C), where each pair represents mutations in the first Fc
polypeptide and second Fc polypeptide. In particular embodiments, a
provided construct contains a first and second Fc polypeptide
containing the pair of mutations L368D/K370S and S364K and
E357Q.
[0279] An additional mechanism that can be used in the generation
of heterodimers is sometimes referred to as "electrostatic
steering" as described in Gunasekaran et al., J. Biol. Chem.
285(25):19637 (2010). This is sometimes referred to herein as
"charge pairs". In this embodiment, electrostatics are used to skew
the formation towards heterodimerization. As those in the art will
appreciate, these may also have an effect on pI, and thus on
purification, and thus could in some cases also be considered pI
variants. However, as these were generated to force
heterodimerization and were not used as purification tools, they
are classified as "steric variants". In one embodiments, a first Fc
polypeptide can contain mutations D221E/P228E/L368E and a second Fc
polypeptide can contain mutations D221R/P228R/K409R. In another
embodiments, a first Fc polypeptide can contain mutations
C220E/P228E/368E and a second Fc polypeptide can contain mutations
C220R/E224R/P228R/K409R.
[0280] In some embodiments, heterodimerization can be facilitated
by pI variants. In some aspects, a pI variant can include those
that increase the pI of the protein (basic changes). In other
aspects, the pI variant can include those that decrease the pI of
the protein (acidic changes). In some cases, all combinations of
these variants can be done, including combinations in which one Fc
polypeptide may be wild type, or a variant that does not display a
significantly different p1 from wild-type, and the other Fc
polypeptide can be either more basic or more acidic. Alternatively,
each Fc polypeptide can be changed, one to more basic and one to
more acidic. In some embodiments, at least one Fc polypeptide is a
negative pI variant Fc containing mutations
Q295E/N384D/Q418E/N421D.
[0281] In some embodiments, a combination of steric
heterodimerization variants (e.g. knob and hole) and pI or charge
pair variants can be used.
[0282] In particular embodiments, the provided constructs contains
(a) a first Fc polypeptide comprising the skew variants
S364K/E357Q; and b) a second Fc polypeptide containing skew
variants L368D/K370S and the pI variants
N208D/Q295E/N384D/Q418E/N421D. In some embodiments, one or both of
the first and second polypeptide can contain further mutations to
reduce Fc effector activity, such as the exemplary mutations
E233P/L234V/L235A/G236del/S267K. An example of such a first Fc
polypeptide and a second Fc polypeptide able to mediate Fc
heterodimeriztion comprise the sequences set forth in SEQ ID
NOs:194 and 195. The first and second Fc polypeptide can be
formatted on either polypeptide chain of the construct.
[0283] The resulting multispecific polypeptide constructs can be
purified by any suitable method such as, for example, by affinity
chromatography over Protein A or Protein G columns. Where two
nucleic acid molecules encoding different polypeptides are
transformed into cells, formation of homo- and heterodimers will
occur. Conditions for expression can be adjusted so that
heterodimer formation is favored over homodimer formation.
[0284] Techniques for recovery of heterodimers from homodimers
based on a differential affinity of the heterodimers for an
affinity reagent are known. In some aspects, such techniques
include designing a heterodimer so that one of the Fc polypeptide
chains does not bind to the affinity reagent protein A. In some
cases, one of the polypeptide chain can contain one or more amino
acid substitution to abrogate or reduce affinity for the protein A
reagent in one of the polypeptides of the Fc heterodimer, see e.g.
WO2017134440, WO2010151792, Jendeberg et al. (Jendeberg et al.,
(1997) J. Immunol. Methods, 201(1): 25-34. In some of these
embodiments, the Fc region may be modified at the protein-A binding
site on one member of the heterodimer so as to prevent protein-A
binding and thereby enable more efficient purification of the
heterodimeric fusion protein. An exemplary modification within this
binding site is Ile253, for example Ile253Arg (I253R). In some
embodiments, the modification may be H435R or H435R/Y436F. In some
embodiments, an Fc polypeptide of an Fc heterodimer can contain a
modification so that it is capable of binding protein A but not
protein G (pA+/pG-). Exemplary pA+/pG- amino acid modifications
include an Fc containing serine at position 428, serine at position
434 and optionally histidine at position 436, with reference to
human IgG1 or comprising these residues at the corresponding
positions in human IgG 2, 3, or 4. In some aspects, such amino acid
modifications in one IgG Fc polypeptide at positions 428, 434 and
optionally 436 reduces or prevents the binding of protein G,
enhancing the purification of the protein.
[0285] In some embodiments, any of such modifications to confer
differential affinity to an affinity reagent can be combined with
any one or more other amino acid modifications described above. For
example, the I253R modification maybe combined with either the
T366S/L368A/Y407V modifications or with the T366W modifications.
The T366S/L368A/Y407V modified Fc is capable of forming homodimers
as there is no steric occlusion of the dimerization interface as
there is in the case of the T336W modified Fc. Therefore, in some
embodiments, the I253R modification is combined with the
T366S/L368A/Y407V modified Fc to disallow purification any
homodimeric Fc that may have formed. Similar modifications can be
employed by combining T366S/L368A/Y407V and H453R.
[0286] In some embodiments, the Fc regions of the heterodimeric
molecule additionally can contain one or more other Fc mutation,
such as any described above. In some embodiments, the heterodimer
molecule contains an Fc region with a mutation that reduces
effector function.
[0287] In some embodiments, one Fc polypeptide of a heterodimeric
Fc comprises the sequence of amino acids set forth in any of SEQ ID
NOS:201 (e.g. SEQ ID NO:82), 86, 207 (e.g. SEQ ID NO:94), or 96,
and the other Fc polypeptide of the heterodimeric Fc contains the
sequence of amino acids set forth in any of SEQ ID NOS:201 (e.g.
SEQ ID NO:83), 87, 205 (e.g. SEQ ID NO:90), 92, 209 (e.g. SEQ ID
NO:98), or 100. In some embodiments, one Fc polypeptide of a
heterodimeric Fc comprises the sequence of amino acids set forth in
any of SEQ ID NOS: 203 (e.g. SEQ ID NO:84), 88, 208 (e.g. SEQ ID
NO:95), or 97 and the other Fc polypeptide of the heterodimeric Fc
comprises the sequence of amino acids set forth in any of SEQ ID
NOS: 204 (e.g. SEQ ID NO:85), 89, 206 (e.g. SEQ ID NO:91), 93, 210
(e.g. SEQ ID NO:99), or 101.
[0288] In some embodiments, the human IgG Fc region is modified to
prevent dimerization. In these embodiments, the fusion proteins of
the present disclosure are monomeric. For example modification at
residue Thr366 to a charged residue, e.g. Thr366Lys, Thr366Arg,
Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E,
respectively), prevents CH3-CH3 dimerization.
[0289] In some embodiments, the Fc region of the fusion protein is
altered at one or more of the following positions to reduce Fc
receptor binding: Leu 234 (L234), Leu235 (L235), Asp265 (D265),
Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) orAla327
(A327). For example, Leu 234Ala (L234A), Leu235Ala (L235A),
Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala
(N297A), Asn325Glu (N325E) orAla327Ser (A327S). In preferred
embodiments, modifications within the Fc region reduce binding to
Fc-receptor-gamma receptors while have minimal impact on binding to
the neonatal Fc receptor (FcRn).
[0290] In some embodiments, the fusion protein contains a
polypeptide derived from an immunoglobulin hinge region. The hinge
region can be selected from any of the human IgG subclasses. For
example, the fusion protein may contain a modified IgG1 hinge
having the sequence of EPKSSDKTHTCPPC (SEQ ID NO: 7), where in the
Cys220 that forms a disulfide with the C-terminal cysteine of the
light chain is mutated to serine, e.g., Cys220Ser (C220S). In other
embodiments, the fusion protein contains a truncated hinge having a
sequence DKTHTCPPC (SEQ ID NO: 8).
[0291] In some embodiments, the fusion protein has a modified hinge
from IgG4, which is modified to prevent or reduce strand exchange,
e.g., Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID
NO: 9). In some embodiments, the fusion protein contains linker
polypeptides. In other embodiments, the fusion protein contains
linker and hinge polypeptides.
[0292] 3. Linkers
[0293] The provided multispecific polypeptide constructs contain a
linker that joins or couples the first component containing the
immunoglobulin Fc region and the second component containing the
CD3 binding region. In some embodiments, the linker is a
non-cleavable linker. In some embodiments, the linker does not
contain a substrate recognition site that is specifically
recognized for cleavage by the protease. Thus, linkers in the
provided multispecific polypeptide constructs do not include an
amino acid sequence that can serve as a substrate for a protease,
such as an extracellular protease. For example, the non-cleavable
linker does not include a cleavage sequence containing at least one
peptide bond which lies within a cleavable peptide sequence of a
protease.
[0294] In some embodiments, the linker is positioned at the end of
the C-terminal region of the Fc region, such that the Fc region is
N-terminal to the CD3 binding region. Because the provided
multispecific polypeptide constructs are multimers, such as dimers,
the provided constructs include a linker joining the first Fc
polypeptide and a first domain (e.g. VH) of the CD3 binding region
of the first polypeptide and the second Fc polypeptide and second
domain (e.g. VL) of the CD3 binding region of the second
polypeptide. Typically, the linkers present in the first and second
polypeptides of the multispecific polypeptide construct are the
same. Thus, in some embodiments, each domain of the CD3 binding
domain is linked via a linker, such as the same linker, to opposite
polypeptides of the Fc, such as heterodimeric Fc.
[0295] Various polypeptide linkers for use in fusion proteins are
known (see e.g. Chen et al. (2013) Adv. Drug. Deliv. 65:1357-1369;
and International PCT publication No. WO 2014/099997, WO2000/24884;
U.S. Pat. Nos. 5,258,498; 5,525,491; 5,525,491, 6,132,992).
[0296] In some embodiments, the linker is chosen so that, when the
CD3 binding region is joined to the Fc region of the multispecific
polypeptide conjugate, the CD3 binding region is constrained and
not able to, or not substantially able to, bind or engage CD3 on
the surface of a cell, e.g. T cell, upon contact of the
multispecific polypeptide construct with the cell. Various assays
can be employed to assess binding or engagement of CD3 by the
multispecific polypeptide construct, including assays to assess T
cell binding, NFAT activation using a reporter system, cytolytic T
cell activity, cytokine production and/or expression of T cell
activation markers. Exemplary assays are shown in the provided
Examples. Typically, the linker also is one that ensures correct
folding of the polypeptide construct, does not exhibit a charge
that would be inconsistent with the activity or function of the
linked polypeptides or form bonds or other interactions with amino
acid residues in one or more of the domains that would impede or
alter activity of the linked polypeptides. In some embodiment, the
linker is a polypeptide linker. The polypeptide linker can be a
flexible linker or a rigid linker or a combination of both.
[0297] In some aspects, the linker is a short, medium or long
linker. In some embodiments, the linker is up to 40 amino acids in
length. In some embodiments, the linker is up to 25 amino acids in
length. In some embodiments, the linker is at least or is at least
about 2 amino acids in length. In some aspects, a suitable length
is, e.g., a length of at least one and typically fewer than about
40 amino acid residues, such as 2-25 amino acid residues, 5-20
amino acid residues, 5-15 amino acid residues, 8-12 amino acid. In
some embodiments, the linker is from or from about 2 to 24 amino
acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino
acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino
acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino
acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino
acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino
acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino
acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino
acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino
acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino
acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino
acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino
acids, 18 to 20 amino acids or 20 to 24 amino acids. In some
embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19 or 20 amino acids in length.
[0298] In certain aspects, the longer the linker length, the
greater the CD3 binding when the multispecific polypeptide
conjugate is bounds to its antigen, e.g. TAA. Thus, in some
aspects, the linker is greater than 12 amino acids in length, such
as greater than 13, 14, 15, 16, 17 or 18 amino acids in length. In
some embodiments, the linker is 12 to 40 amino acids in length, 12
to 30 amino acids, 12 to 24 amino acids, 12 to 18 acids, 12 to 15
amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 24
amino acids, 15 to 18 amino acids, 18 to 40 amino acids, 18 to 30
amino acids, 18 to 24 amino acids, 24 to 40 amino acids, 24 to 30
amino acids or 30 to 40 amino acids.
[0299] The linkers can be naturally-occurring, synthetic or a
combination of both. Particularly suitable linker polypeptides
predominantly include amino acid residues selected from Glycine
(Gly), Serine (Ser), Alanine (Ala), and Threonine (Thr). For
example, the linker may contain at least 75% (calculated on the
basis of the total number of residues present in the peptide
linker), such as at least 80%, at least 85%, or at least 90% of
amino acid residues selected from Gly, Ser, Ala, and Thr. The
linker may also consist of Gly, Ser, Ala and/or Thr residues only.
In some embodiments, the linker contains 1-25 glycine residues,
5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine
residues. In some aspects, suitable peptide linkers typically
contain at least 50% glycine residues, such as at least 75% glycine
residues. In some embodiments, a peptide linker comprises glycine
residues only. In some embodiments, a peptide linker comprises
glycine and serine residues only.
[0300] In some embodiments, these linkers are composed
predominately of the amino acids Glycine and Serine, denoted as
GS-linkers herein. In some embodiments, the linker contains (GGS)n,
wherein n is 1 to 10, such as 1 to 5, for example 1 to 3, such as
GGS(GGS)n (SEQ ID NO:171), wherein n is 0 to 10. In particular
embodiments, the linker contains the sequence (GGGGS)n (SEQ ID NO:
173), wherein n is 1 to 10 or n is 1 to 5, such as 1 to 3. In
further embodiments, the linker contains (GGGGGS)n (SEQ ID NO:172),
wherein n is 1 to 4, such as 1 to 3. The linker can include
combinations of any of the above, such as repeats of 2, 3, 4, or 5
GS, GGS, GGGGS, and/or GGGGGS linkers may be combined. In some
embodiments, such a linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18 or 19 amino acids in length.
[0301] In some embodiments, the linker is (in one-letter amino acid
code): GGS, GGGGS (SEQ ID NO: 149), or GGGGGS (SEQ ID NO: 135). In
some embodiments, the GS-linker comprises an amino acid sequence of
GGSGGS, i.e., (GGS).sub.2 (SEQ ID NO: 10); GGSGGSGGS, i.e.,
(GGS).sub.3 (SEQ ID NO: 11); GGSGGSGGSGGS, i.e., (GGS).sub.4 (SEQ
ID NO: 12); GGSGGSGGSGGSGGS, i.e., (GGS).sub.5 (SEQ ID NO: 13);
GGGGGSGGGGGSGGGGGS, i.e., (G5S).sub.3 (SEQ ID NO: 119),
GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147) and GGGGSGGGGSGGGGS (SEQ ID
NO:170). In some embodiments, the linker is GGGG (SEQ ID NO:103).
In some embodiments, the linker is GGGGG (SEQ ID NO:192). In some
of any of the above examples, serine can be replaced with alanine
(e.g., (Gly4Ala) or (Gly3Ala)).
[0302] In some embodiments, the linker includes a peptide linker
having the amino acid sequence Gly.sub.xXaa-Gly.sub.y-Xaa-Gly.sub.z
(SEQ ID NO:174), wherein each Xaa is independently selected from
Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile),
Methionine (Met), Phenylalanine (Phe), Tryptophan (Trp), Proline
(Pro), Glycine (Gly), Serine (Ser), Threonine (Thr), Cysteine
(Cys), Tyrosine (Tyr), Asparagine (Asn), Glutamine (Gln), Lysine
(Lys), Arginine (Arg), Histidine (His), Aspartate (Asp), and
Glutamate (Glu), and wherein x, y, and z are each integers in the
range from 1-5. In some embodiments, each Xaa is independently
selected from the group consisting of Ser, Ala, and Thr. In a
specific variation, each of x, y, and z is equal to 3 (thereby
yielding a peptide linker having the amino acid sequence
Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly (SEQ ID NO:175),
wherein each Xaa is selected as above.
[0303] In some embodiments, the linker is serine-rich linkers based
on the repetition of a (SSSSG)n (SEQ ID NO:185) motif where n is at
least 1, though n can be 2, 3, 4, 5, 6, 7, 8 and 9.
[0304] In some cases, it may be desirable to provide some rigidity
into the peptide linker. This may be accomplished by including
proline residues in the amino acid sequence of the peptide linker.
Thus, in some embodiments, a linker comprises at least one proline
residue in the amino acid sequence of the peptide linker. For
example, a peptide linker can have an amino acid sequence wherein
at least 25% (e.g., at least 50% or at least 75%) of the amino acid
residues are proline residues. In one particular embodiment, the
peptide linker comprises proline residues only.
[0305] In some aspects, a peptide linker comprises at least one
cysteine residue, such as one cysteine residue. For example, in
some embodiments, a linker comprises at least one cysteine residue
and amino acid residues selected from the group consisting of Gly,
Ser, Ala, and Thr. In some such embodiments, a linker comprises
glycine residues and cysteine residues, such as glycine residues
and cysteine residues only. Typically, only one cysteine residue
will be included per peptide linker. One example of a specific
linker comprising a cysteine residue includes a peptide linker
having the amino acid sequence Gly.sub.m-Cys-Gly.sub.n, wherein n
and m are each integers from 1-12, e.g., from 3-9, from 4-8, or
from 4-7. In a specific variation, such a peptide linker has the
amino acid sequence GGGGG-C-GGGGG (SEQ ID NO:177).
[0306] In some embodiments, the linker of the fusion protein is a
structured or constrained linker. In particular embodiments, the
structured linker contains the sequence (AP)n or (EAAAK)n (SEQ ID
NO:178), wherein n is 2 to 20, preferably 4 to 10, including but
not limited to, AS-(AP)n-GT (SEQ ID NO:179) or AS-(EAAAK)n-GT (SEQ
ID NO:180), wherein n is 2 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14 or 15. In other embodiments, the linker
comprises the sequences (GGGGA)n (SEQ ID NO:181), (PGGGS)n (SEQ ID
NO:182), (AGGGS)n (SEQ ID NO:183) or GGS-(EGKSSGSGSESKST)n-GGS (SEQ
ID NO:184, wherein n is 2 to 20. In some embodiments, the linker is
SSSASASSA (SEQ ID NO:186), GSPGSPG (SEQ ID NO:187), or ATTTGSSPGPT
(SEQ ID NO:176). In some embodiments, such linkers, by virtue of
their structure, may be more resistant to proteolytic degradation,
thereby offering an advantage when injected in vivo.
[0307] In some embodiments, the linker is not a cleavable linker
(used interchangeably with non-cleavable linker). In some
embodiments, the linker is not cleavable by a protease. In some
embodiments, a linker that is not a cleavable linker or that is not
cleavable by a protease is one that is generally stable for in vivo
delivery or recombinant production. In some aspects, a linker that
is not cleavable by a protease includes those that do not contain
at least one peptide bond which preferably lies within a cleavable
peptide sequence or recognition site of a protease. In particular
embodiments, a non-cleavable linker is not a target substrate for a
protease, such that it is not preferentially or specifically
cleaved by a protease compared to a linker that contains a
substrate recognition site for the same protease.
[0308] In some embodiments, the linker does not contains a
substrate recognition site or cleavage site for a particular
protease, which is the sequence recognized by the active site of a
protease that is cleaved by a protease. Typically, for example, for
a serine protease, a cleavage sequence is made up of the P1-P4 and
P1'-P4' amino acids in a substrate, where cleavage occurs after the
P1 position. Typically, a cleavage sequence for a serine protease
is six residues in length to match the extended substrate
specificity of many proteases, but can be longer or shorter
depending upon the protease. Typically, the linker does not include
a P1-P1' scissile bond sequence that is recognized by a
protease.
[0309] In some aspects, a non-cleavable linker or a linker that
does not contain a substrate recognition site that is specifically
recognized for cleavage by a protease is one whose cleavage by a
protease is substantially less than cleavage of a target substrate
of the protease. Typically, a protease exhibits specificity or
preference for cleavage of a particular target substrate compared
to another non-target substrate. Such a degree of specificity can
be determined based on the rate constant of cleavage of a sequence,
e.g. linker sequence, which is a measure of preference of a
protease for its substrate and the efficiency of the enzyme. Any
method to determine the rate of increase of cleavage over time in
the presence of various concentrations of substrate can be used to
calculate the specificity constant. For example, a substrate is
linked to a fluorogenic moiety, which is released upon cleavage by
a protease. By determining the rate of cleavage at different
protease concentrations the specificity constant for cleavage
(k.sub.cat/K.sub.m) can be determined for a particular protease
towards a particular linker. In some embodiments, a non-cleavable
linker, or a linker that does not contain a substrate recognition
site that is specifically recognized for cleavage by a protease, is
a linker that, if cleaved at all, is cleaved by a protease at a
rate of less than 1.times.10.sup.4 M.sup.-1S.sup.-, or less than
5.times.10.sup.3 M.sup.-1S, less than 1.times.10.sup.3 M.sup.-1S,
or less than 1.times.10.sup.2 M.sup.-1S or less.
[0310] In some embodiments, the linkers in the multispecific
constructs provided herein do not contain a substrate recognition
site for a protease that include, for example, matrix
metalloproteases (MMP), cysteine proteases, serine proteases and
plasmin activators. In particular embodiments, the linker does not
contain a substrate recognition site for a protease that is a
protease that is produced by a tumor, an activated immune effector
cell (e.g. a T cell or a NK cell), or a cell in a tumor
microenvironment.
[0311] In some embodiments, the linker does not contain a substrate
recognition site that is specifically recognized by one or more of
the following enzymes or proteases: ADAMS, ADAMTS, e.g. ADAMS;
ADAMS; ADAM10; ADAM12; ADAM15; ADAM17/TACE; ADAMDEC1; ADAMTS1;
ADAMTS4; ADAMTSS; aspartate proteases, e.g., BACE or Renin;
aspartic cathepsins, e.g., Cathepsin D or Cathepsin E; Caspases,
e.g., Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5,
Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, or Caspase
14; cysteine cathepsins, e.g., Cathepsin B, Cathepsin C, Cathepsin
K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P;
Cysteine proteinases, e.g., Cruzipain; Legumain; Otubain-2; KLKs,
e.g., KLK4, KLKS, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14;
Metallo proteinases, e.g., Meprin; Neprilysin; PSMA; BMP-1; MMPs,
e.g., MMP1, MMP2, MMP3, MMP1, MMP8, MMP9, MMP10, MMP11, MMP12,
MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24,
MMP26, or MMP27, serine proteases, e.g., activated protein C,
Cathepsin A, Cathepsin G, Chymase, coagulation factor proteases
(e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), Elastase, Granzyme B,
Guanidinobenzoatase, HtrA1, Human Neutrophil Elastase, Lactoferrin,
Marapsin, NS3/4A, PACE4, Plasmin, PSA, tPA, Thrombin, Tryptase,
uPA; Type II Transmembrane Serine Proteases (TTSPs), e.g., DESC1,
DPP-4, FAP, Hepsin, Matriptase-2, Matriptase, TMPRSS2, TMPRSS3, or
TMPRSS4; and any combination thereof. In some embodiments, the
linker does not contain a substrate recognition site that is
specifically recognized by granzyme B, a matriptase or an MMP, such
as MMP-2.
[0312] In some embodiments, the linker does not comprise an amino
acid that is a substrate for Granzyme B. In some embodiments, the
linker does not contain an amino acid sequence having the general
formula P4 P3 P2 P1.dwnarw.P1' (SEQ ID NO: 150), wherein P4 is
amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E,
D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino
acid D or E; and P1' is amino acid I, L, Y, M, F, V, T, S, G or A.
In some embodiments, the linker does not contain an amino acid
sequence having the general formula P4 P3 P2 P1.dwnarw.P1' (SEQ ID
NO: 151), wherein P4 is amino acid I or L; P3 is amino acid E; P2
is amino acid P or A; P1 is amino acid D; and P1' is amino acid I,
V, T, S, or G.
[0313] In some embodiments, the linker does not contain the amino
acid sequence LEAD (SEQ ID NO: 22), LEPD(SEQ ID NO: 142), or LEAE
(SEQ ID NO:143). In some embodiments, the linker does not contain
the amino acid sequence IEPDI (SEQ ID NO:136), LEADT (SEQ ID
NO:137), IEPDG (SEQ ID NO:138), IEPDV (SEQ ID NO:139), IEPDS (SEQ
ID NO:140), IEPDT (SEQ ID NO:141), IEPDP (SEQ ID NO:144), LEPDG
(SEQ ID NO:152) or LEADG (SEQ ID NO:153).
[0314] In some embodiments, the linker does not comprise an amino
acid that is a substrate for matriptase. In some embodiments, the
linker does not comprises the sequence P1QAR.dwnarw.(A/V) (SEQ ID
NO: 154), wherein P1 is any amino acid. In some embodiments, the
linker does not comprises the sequence RQAR(A/V) (SEQ ID NO: 155).
In some embodiments, the linker does not comprise the amino acid
sequence RQAR (SEQ ID NO: 23). In some embodiments, the linker does
not comprise the amino acid sequence RQARV (SEQ ID NO: 156)
[0315] In some embodiments, the linker does not comprise an amino
acid that is a substrate for one or more matrix metalloproteases
(MMPs). In some embodiments, the MMP is MMP-2. In some embodiments,
the linker does not contain a sequence having the general formula
P3 P2 P1.dwnarw.P1' (SEQ ID NO: 157), wherein P3 is P, V or A; P2
is Q or D; P1 is A or N; and P1' is L, I or M. In some embodiments,
the linker does not contain the general formula P3 P2 P1.dwnarw.P1'
(SEQ ID NO: 158), wherein P3 is P; P2 is Q or D; P1 is A or N; and
P1' is L or I. In some embodiments, the linker does not comprise
the amino acid sequence PAGL (SEQ ID NO: 24).
[0316] In some embodiments, the linker is not a linker comprising
the amino acid sequence set forth as TGLEADGSPAGLGRQARVG (SEQ ID
NO: 25); TGLEADGSRQARVGPAGLG (SEQ ID NO: 26); TGSPAGLEADGSRQARVGS
(SEQ ID NO: 27); TGPAGLGLEADGSRQARVG (SEQ ID NO: 28);
TGRQARVGLEADGSPAGLG (SEQ ID NO: 29); TGSRQARVGPAGLEADGS (SEQ ID NO:
30); and TGPAGLGSRQARVGLEADGS (SEQ ID NO: 31); GPAGLGLEPDGSRQARVG
(SEQ ID NO: 104); GGSGGGGIEPDIGGSGGS (SEQ ID NO: 105);
GGSGGGGLEADTGGSGGS (SEQ ID NO: 106); GSIEPDIGS (SEQ ID NO: 107);
GSLEADTGS (SEQ ID NO: 108); GGSGGGGIEPDGGGSGGS (SEQ ID NO: 109);
GGSGGGGIEPDVGGSGGS (SEQ ID NO: 110); GGSGGGGIEPDSGGSGGS (SEQ ID NO:
111); GGSGGGGIEPDTGGSGGS (SEQ ID NO: 112); GGGSLEPDGSGS (SEQ ID NO:
113); and GPAGLGLEADGSRQARVG (SEQ ID NO: 114), GGEGGGGSGGSGGGS (SEQ
ID NO: 115); GSSAGSEAGGSGQAGVGS (SEQ ID NO: 116);
GGSGGGGLEAEGSGGGGS (SEQ ID NO: 117); GGSGGGGIEPDPGGSGGS(SEQ ID NO:
118); TGGSGGGGIEPDIGGSGGS (SEQ ID NO: 148).
[0317] 4. Antigen Binding Domains:
[0318] The multispecific polypeptide constructs of the present
disclosure include at least one antigen binding domain, such as at
least a first antigen binding domain and a second antigen binding
domain. In some aspects, the antigen binding domain, or
independently each of the antigen binding domains, is selected from
an antibody or antigen binding fragment, a natural cognate binding
partner, an Anticalin (engineered lipocalin), a Darpin, a Fynomer,
a Centyrin (engineered fibroneticin III domain), a cystine-knot
domain, an Affilin, an Affibody, or an engineered CH3 domain. In
some embodiments, the natural cognate binding partner comprises an
extracellular domain or binding fragment thereof of the native
cognate binding partner of the TAA, or a variant thereof that
exhibits binding activity to the TAA.
[0319] In some embodiments, a TAA is a counter-structure that is
present primarily on tumor cells of a mammalian subject but
generally not found on normal cells of the mammalian subject. A
tumor specific antigen need not be exclusive to tumor cells but the
percentage of cells of a particular mammal that have the tumor
associated antigen is sufficiently high or the levels of the tumor
associated antigen on the surface of the tumor are sufficiently
high such that it can be targeted by anti-tumor therapeutics, such
as multispecific polypeptide constructs as provided, and provide
prevention or treatment of the mammal from the effects of the
tumor. In some embodiments, in a random statistical sample of cells
from a mammal with a tumor, at least 50% of the cells displaying a
TAA are cancerous. In other embodiments, at least 60%, 70%, 80%,
85%, 90%, 95%, or 99% of the cells displaying a TAA are
cancerous.
[0320] In some embodiments, the antigen binding domain, or
independently each of the antigen binding domains, such as the
first antigen-binding domain and the second antigen binding
domains, includes one or more copies of an antibody or an
antigen-binding fragment thereof. In some embodiments, the antigen
binding domain, or independently each of the antigen binding
domains, includes one or more copies of an antibody or an
antigen-binding fragment thereof selected from the group consisting
of a Fab fragment, a F(ab').sub.2 fragment, an Fv fragment, a scFv,
a scAb, a dAb, a single domain heavy chain antibody, and a single
domain light chain antibody. In some embodiments, the antigen
binding domain, or independently each of the antigen binding
domains, such as the first antigen-binding domain and the second
antigen binding domains, is a single chain antibody. In some
examples, the single chain is an scFv, a scAb, a single domain
heavy chain antibody, or a single domain light chain antibody. In
some embodiments, each of the first antigen-binding domain and the
second antigen binding domains includes one or more single domain
antibody (sdAb) fragments, for example V.sub.HH, V.sub.NAR,
engineered V.sub.H or V.sub.K domains. V.sub.HHs can be generated
from natural camelid heavy chain only antibodies, genetically
modified rodents that produce heavy chain only antibodies, or
naive/synthetic camelid or humanized camelid single domain antibody
libraries. V.sub.NARs can be generated from cartilaginous fish
heavy chain only antibodies. Various methods have been implemented
to generate monomeric sdAbs from conventionally heterodimeric
V.sub.H and V.sub.K domains, including interface engineering and
selection of specific germline families.
[0321] In some embodiments, the antigen binding domain or
independently each of the antigen binding domains, such as the
first antigen-binding domain and/or the second antigen binding
domains, of the multispecific polypeptide constructs contains at
least one sdAb or an scFv that binds a TAA. In some embodiments,
the at least one scFv or sdAb that binds a TAA is positioned
amino-terminally relative to the Fc region and/or
carboxy-terminally relative to the CD3 binding region of the
multispecific polypeptide construct. In some embodiments, the
multispecific polypeptide construct contains only one scFv or sdAb
that binds to a TAA, which can be positioned either
amino-terminally relative to the Fc region and/or
carboxy-terminally relative to the CD3 binding region. In some
embodiments, the multispecific polypeptide construct contains two
scFvs or sdAbs that bind to a TAA, positioned amino-terminally
relative to the Fc region and/or carboxy-terminally relative to the
CD3 binding region. In some embodiments, the multispecific
polypeptide construct contains three scFv or sdAb, in which two are
positioned amino-terminally relative to the Fc region or
carboxy-terminally relative to the CD3 binding region, and the
third is positioned at the other end of the multispecific
polypeptide construct.
[0322] In some embodiments, the multispecific polypeptide construct
is formed from or includes two polypeptides, including a first
polypeptide comprising a first Fc polypeptide of a heterodimeric Fc
region, a linker, a VH domain of an anti-CD3 antibody or antigen
binding fragment (e.g. Fv), and an scFv or sdAb that binds to a
tumor-associated antigen; and a second polypeptide comprising a
second Fc polypeptide of the heterodimeric Fc region, the linker, a
VL domain of the anti-CD3 antibody or antigen binding fragment
(e.g. Fv) and, optionally, the same or different scFv or sdAb that
binds to a tumor-associated antigen. The scFv or sdAb that binds to
a TAA can be positioned amino terminally relative to an Fc
polypeptide of the heterodimeric Fc and/or carboxy-terminally
relative to a VH or VL chain of the CD3 binding region. In some
embodiments, the antigen binding domain, or independently each of
the antigen binding domains, of the multispecific polypeptide
constructs contains VH and VL sequences assembled as FABs or scFvs.
In some embodiments, the antigen binding domain, or independently
each of the antigen binding domains, of the multispecific
polypeptide constructs contains binding domains as single domain
antibodies (sdAbs).
[0323] In some embodiments, the antigen binding domain or
independently each of the antigen binding domains, such as the
first antigen-binding domain and the second antigen binding
domains, contains more than one chain. In some embodiments, the
antigen binding domain or independently each of the antigen binding
domains, such as the first antigen-binding domain and/or the second
antigen binding domains, of the multispecific polypeptide
constructs contains VH and VL sequences assembled as FABs.
[0324] In some embodiments, the antigen binding domain or
independently each of the antigen binding domains, such as the
first antigen-binding domain and/or the second antigen binding
domains, of the multispecific polypeptide constructs contains a
VH-CH1 (Fd) and a VL-CL of a Fab antibody that binds a TAA. In some
embodiments, the Fab antibody containing a VH-CH1 (Fd) and a VL-CL
is positioned amino-terminally relative to the Fc region and/or
carboxy-terminally relative to the CD3 binding region of the
multispecific polypeptide construct. In some embodiments, the
multispecific polypeptide construct contains only one Fab antibody,
containing a VH-CH1 (Fd) and VL-CL, that binds to a TAA, which can
be positioned either amino-terminally relative to the Fc region
and/or carboxy-terminally relative to the CD3 binding region. In
some embodiments, the multispecific polypeptide construct contains
two Fab antibody fragments, each containing a VH-CH1 (Fd) and
VL-CL, that binds to a TAA, in which one is positioned
amino-terminally relative to the Fc region and the other is
positioned carboxy-terminally relative to the CD3 binding
region.
[0325] In some embodiments, the multispecific polypeptide construct
is formed from or includes three or more polypeptides, including a
first polypeptide comprising a first Fc polypeptide of a
heterodimeric Fc region, a linker and a VH-CH1 (Fd) or VL-CL of a
Fab antibody fragment that binds to a tumor-associated antigen; a
second polypeptide comprising a second Fc polypeptide of the
heterodimeric Fc region, the linker and, optionally, the same
VH-CH1 (Fd) or VL-CL of the Fab antibody fragment that binds to a
tumor-associated antigen, and a third polypeptide comprising the
other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment that
binds to the TAA.
[0326] In some embodiments, the antigen binding domain, or
independently each of the antigen binding domains, is or includes
an extracellular domain or binding fragment thereof of the native
cognate binding partner of the TAA, or a variant thereof that
exhibits binding activity to the TAA.
[0327] In some embodiments, each of the antigen binding domains,
such as each of the first antigen-binding domain and the second
antigen binding domains, bind the same antigen. In some
embodiments, each of the first antigen-binding domain and the
second antigen binding domains bind a different antigen. In some
embodiments, each of the antigen binding domains, such as each of
the first antigen-binding domain and the second antigen binding
domains, bind the same tumor associated antigen (TAA). In some
embodiments, each of the antigen binding domains, such as each of
the first antigen-binding domain and the second antigen binding
domains, bind a different TAA. In some embodiments, each of the
antigen binding domains, such as each of the first antigen-binding
domain and the second antigen binding domains, bind a different
epitope on the same TAA. In some embodiments, each of the antigen
binding domains, such as each of the first antigen-binding domain
and the second antigen binding domains, bind the same epitope on
the same TAA.
[0328] In some embodiments, the antigen binding domains results in
monovalent, bivalent, trivalent, or tetravalent binding to the TAA.
In some embodiments, bivalent binding to the TAA comprises two
antigen binding domains that bind the same epitope of the same
antigen (e.g. mono-epitopic). In some embodiments, bivalent binding
to the TAA comprises two antigen binding domains that bind
different epitopes of the same antigen (e.g. bi-epitopic). In some
embodiments, monovalent binding to the TAA comprises one antigen
binding domain that binds one epitope of the antigen (e.g.
mono-epitopic).
[0329] In some embodiments, the TAA is selected from the group
consisting of 1-92-LFA-3, 5T4, Alpha-4 integrin, Alpha-V integrin,
alpha4beta1 integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y,
Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement,
C-242, CA9, CA19-9, (Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6,
CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33,
CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56,
CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123,
CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248,
CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet,
Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13,
CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1,
EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3,
EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8,
FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa),
GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR,
GP IIb/IIIa receptors, Gp130, GPIIB/IIIA, GPNMB, GRP78, HER2/neu,
HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS, IFNalpha, IFNbeta,
IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2,
IL11, IL12, IL12p40, IL-12R, IL-12Rbeta1, IL13, IL13R, IL15, IL17,
IL18, IL21, IL23, IL23R, IL27/IL27R (wsx1), IL29, IL-31R,
IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin Receptor, Jagged
Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT,
LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, Mesothelin, MRP4, MUC1, Mucin-16
(MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, Notch Receptors,
Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2,
PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2,
Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4,
Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8,
TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31,
TNFalpha, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin,
Transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF,
VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA,
WISP-1, WISP-2, and WISP-3.
[0330] In some embodiments, at least one antigen binding domain, or
independently each antigen binding domain, binds the tumor
associated antigen (TAA) folate receptor alpha (FRa). For example,
the antigen binding domain contains the binding domain as an sdAb
that binds FR.alpha.. Exemplary FR.alpha.-binding sdAbs are set
forth in SEQ ID NOS: 120, 121, and 122.
[0331] In some embodiments, at least one antigen binding domain, or
independently each antigen binding domain, binds the tumor
associated antigen (TAA) cMET. For example, the antigen binding
domain contains the binding domain as a sdAb that binds cMET. An
exemplary cMET-binding sdAb is set forth in SEQ ID NO: 123 (U.S.
Pat. No. 9,346,884).
[0332] In some embodiments, at least one antigen binding domain, or
independently each antigen binding domain, binds the tumor
associated antigen (TAA) B7H3. For example, the antigen binding
domain contains the binding domain as an scFv that binds B7H3. An
exemplary B7H3-binding scFv is set forth in SEQ ID NO: 124. In some
embodiments, the antigen binding domain is a sdAb, such as a VHH.
Exemplary B7H3-binding sdAbs are set forth in any of SEQ ID NOS:
214-218. In some embodiments, the antigen binding domain is or
contains a Fab antibody fragment comprising a VH-CH1 (Fd) and LC.
An exemplary B7H3 Fd is set forth in SEQ ID NO: 127 and an
exemplary B7H3 LC is set forth in SEQ ID NO: 128 (PCT Publication
No, WO2017/030926).
[0333] In some embodiments, at least one antigen binding domain, or
independently each antigen binding domain, binds the tumor
associated antigen (TAA) CD20. In some embodiments, such an
antigen-binding domain contains a VH set forth in SEQ ID NO: 189
and a VL set forth in SEQ ID NO: 190 or a sequence that exhibits at
least at or about 85%, 90%, 95%, 96%, 97%, 98%, 98%, or 99%
sequence identity to SEQ ID NO: 189 or SEQ ID NO:190. For example,
the antigen binding domain contains the binding domain as an scFv
that binds CD20. Exemplary CD20-binding scFvs are set forth in SEQ
ID NO: 125and 213 (U.S. Pub. No. US 2005/0123546).
[0334] In some embodiments, at least one antigen binding domain, or
independently each antigen binding domain, binds the tumor
associated antigen (TAA) DLL3. For example, the antigen binding
domain contains the binding domain as an scFv that binds DLL3.
Exemplary DLL3-binding scFv is set forth in SEQ ID NO: 126 and 188
(U.S. Pub. No. US 2017/0037130). In some embodiments, the antigen
binding domain is a sdAb, such as a VHH. Exemplary DLL3-binding
sdAbs are set forth in any of SEQ ID NO: 219 or SEQ ID NO:220. In
some embodiments, the antigen binding domain is or contains a Fab
antibody fragment comprising a Fd and LC that binds DLL3. An
exemplary DLL3 Fd is set forth in SEQ ID NO: 133 and an exemplary
DLL3 LC is set forth in SEQ ID NO: 134 (U.S. Pat. No.
8,044,178).
[0335] In some embodiments, at least one antigen binding domain, or
independently each antigen binding domain, binds the tumor
associated antigen (TAA) 5T4. An exemplary 5T4 Fd is set forth in
SEQ ID NO: 129 and an exemplary 5T4 LC is set forth in SEQ ID NO:
130. In some embodiments, the antibody binding domain comprises a
VH-CH1 (Fd) or VL-CL as set forth in SEQ ID NOS: 167 and 168 (U.S.
Pat. No. 8,044,178).
[0336] In some embodiments, at least one antigen binding domain, or
independently each antigen binding domain, binds the tumor
associated antigen (TAA) gpNMB. In some embodiments, the antigen
binding domain is or contains a Fab fragment comprising a Fd and LC
chain. An exemplary gpNMB Fd is set forth in SEQ ID NO: 131 and an
exemplary gpNMB LC is set forth in SEQ ID NO: 132.
[0337] In some embodiments, the antigen binding domain is linked,
directly or indirectly via a linker, to the Fc region and/or to the
CD3 binding region. In some embodiments, linkage is via a linker.
In some embodiments, the linker is a linking peptide (LP), which
can include any flexible or rigid linker as described in Section
11.3, although generally peptides linking the antigen binding
domain or domains is not a cleavable linker.
[0338] In some embodiments, the multispecific polypeptide construct
comprises a first linking peptide (LP1) between the first antigen
binding domain and the Fc region. In some embodiments, the
multispecific polypeptide construct comprises a second linking
peptide (LP2) between the CD3 binding region and the second antigen
binding domain. In some embodiments, the multispecific polypeptide
construct comprises a first linking peptide (LP1) between the first
antigen binding domain and the Fc region and a second linking
peptide (LP2) between the CD3 binding region and the second antigen
binding domain. In some aspects, the multispecific polypeptide
construct has the structural arrangement from N-terminus to
C-terminus as follows: first antigen binding domain-LP1-Fc
region-linker-CD3 binding region-LP2-second antigen binding domain.
In some embodiments, the two linking peptides are not identical to
each other.
[0339] In some embodiments, the LP1 or LP2 is independently a
peptide of about 1 to 20 amino acids in length. In some
embodiments, the LP1 or LP2 is independently a peptide that is or
comprises any Gly-Ser linker as set forth in SEQ ID NOs: 10-13,
119, 135, 147, 149 or GGS.
III. PHARMACEUTICAL COMPOSITION
[0340] Provided herein are compositions of any of the provided
multispecific polypeptide constructs. It will be appreciated that
administration of therapeutic entities in accordance with the
disclosure will be administered with suitable carriers, excipients,
and other agents that are incorporated into formulations to provide
improved transfer, delivery, tolerance, and the like. A multitude
of appropriate formulations can be found in the formulary known to
all pharmaceutical chemists: Remington's Pharmaceutical Sciences
(15th ed., Mack Publishing Company, Easton, Pa. (1975)),
particularly Chapter 87 by Blaug, Seymour, therein. These
formulations include, for example, powders, pastes, ointments,
jellies, waxes, oils, lipids, lipid (cationic or anionic)
containing vesicles (such as Lipofectin.TM.), DNA conjugates,
anhydrous absorption pastes, oil-in-water and water-in-oil
emulsions, emulsions carbowax (polyethylene glycols of various
molecular weights), semi-solid gels, and semi-solid mixtures
containing carbowax. Any of the foregoing mixtures may be
appropriate in treatments and therapies in accordance with the
present disclosure, provided that the active ingredient in the
formulation is not inactivated by the formulation and the
formulation is physiologically compatible and tolerable with the
route of administration. See also Baldrick P. "Pharmaceutical
excipient development: the need for preclinical guidance." Regul.
Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and
development of solid protein pharmaceuticals." Int. J. Pharm.
203(1-2):1-60 (2000), Charman WN "Lipids, lipophilic drugs, and
oral drug delivery-some emerging concepts." J Pharm
Sci.89(8):967-78 (2000), Powell et al. "Compendium of excipients
for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311
(1998) and the citations therein for additional information related
to formulations, excipients and carriers well known to
pharmaceutical chemists.
[0341] In some embodiments, the multispecific polypeptide
constructs, conjugated multispecific polypeptide constructs, and
compositions thereof--referred to collectively herein as the
Therapeutic(s) and derivatives, fragments, analogs and homologs
thereof, can be incorporated into pharmaceutical compositions
suitable for administration. Principles and considerations involved
in preparing such compositions, as well as guidance in the choice
of components are provided, for example, in Remington's
Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th
ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton,
Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities,
Limitations, And Trends, Harwood Academic Publishers, Langhorne,
Pa., 1994; and Peptide And Protein Drug Delivery (Advances In
Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
[0342] Such compositions typically comprise the multispecific
polypeptide construct or a conjugated thereof and a
pharmaceutically acceptable carrier. Where a multispecific
polypeptide construct includes a fragment of an antibody, the
smallest fragment of the antibody that specifically binds to the
target protein can be used. For example, based upon the
variable-region sequences of an antibody, peptide molecules can be
designed that retain the ability of the antibody to bind the target
protein sequence. Such peptides can be synthesized chemically
and/or produced by recombinant DNA technology. (See, e.g., Marasco
et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
[0343] As used herein, the term "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Suitable carriers are described in
the most recent edition of Remington's Pharmaceutical Sciences, a
standard reference text in the field, which is incorporated herein
by reference. Suitable examples of such carriers or diluents
include, but are not limited to, water, saline, ringer's solutions,
dextrose solution, and 5% human serum albumin. Liposomes and
non-aqueous vehicles such as fixed oils may also be used. The use
of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated.
[0344] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0345] A pharmaceutical composition of the disclosure is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0346] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
suitable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, aluminum monostearate and
gelatin.
[0347] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. Where the composition is
lyophilized, sterilization using this method may be conducted
either prior to or following lyophilization and reconstitution. The
composition for parenteral administration may be stored in
lyophilized form or in solution. In addition, parenteral
compositions generally are placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[0348] In some embodiments, the pharmaceutical composition is
administered to a subject through any route, including orally,
transdermally, by inhalation, intravenously, intra-arterially,
intramuscularly, direct application to a wound site, application to
a surgical site, intraperitoneally, by suppository, subcutaneously,
intradermally, transcutaneously, by nebulization, intrapleurally,
intraventricularly, intra-articularly, intraocularly, or
intraspinally.
[0349] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0350] For administration by inhalation, the multispecific
polypeptide construct are delivered in the form of an aerosol spray
from pressured container or dispenser that contains a suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[0351] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0352] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0353] In one embodiment, the Therapeutics are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as sustained/controlled release formulations,
including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic acid. Methods for preparation of
such formulations will be apparent to those skilled in the art.
[0354] For example, the Therapeutics can be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions.
[0355] In some embodiments, the pharmaceutical composition
comprises a pharmaceutically-acceptable excipient, for example a
filler, binder, coating, preservative, lubricant, flavoring agent,
sweetening agent, coloring agent, a solvent, a buffering agent, a
chelating agent, or stabilizer. Examples of
pharmaceutically-acceptable fillers include cellulose, dibasic
calcium phosphate, calcium carbonate, microcrystalline cellulose,
sucrose, lactose, glucose, mannitol, sorbitol, maltol,
pregelatinized starch, corn starch, or potato starch. Examples of
pharmaceutically-acceptable binders include polyvinylpyrrolidone,
starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose,
polyethylene glycol, methyl cellulose, or cellulose. Examples of
pharmaceutically-acceptable coatings include hydroxypropyl
methylcellulose (HPMC), shellac, corn protein zein, or gelatin.
Examples of pharmaceutically-acceptable disintegrants include
polyvinylpyrrolidone, carboxymethyl cellulose, or sodium starch
glycolate. Examples of pharmaceutically-acceptable lubricants
include polyethylene glycol, magnesium stearate, or stearic acid.
Examples of pharmaceutically-acceptable preservatives include
methyl parabens, ethyl parabens, propyl paraben, benzoic acid, or
sorbic acid. Examples of pharmaceutically-acceptable sweetening
agents include sucrose, saccharine, aspartame, or sorbitol.
Examples of pharmaceutically-acceptable buffering agents include
carbonates, citrates, gluconates, acetates, phosphates, or
tartrates.
[0356] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. In some embodiments, the pharmaceutical composition
further comprises an agent for the controlled or sustained release
of the product, such as injectable microspheres, bio-erodible
particles, polymeric compounds (polylactic acid, polyglycolic
acid), beads, or liposomes. Examples of sustained-release matrices
include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0357] The materials can also be obtained commercially from Alza
Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions
(including liposomes targeted to infected cells with monoclonal
antibodies to viral antigens) and can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0358] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the disclosure are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0359] Further provided are kits comprising the pharmaceutical
compositions (or articles of manufacture) described herein. The
pharmaceutical compositions can be included in a container, pack,
or dispenser together with instructions for administration. The
kits described herein may also include other materials desirable
from a commercial and user standpoint, including other buffers,
diluents, filters, needles, syringes, and package inserts with
instructions for performing any methods described herein.
[0360] The formulation can also contain more than one multispecific
polypeptide construct as necessary for the particular indication
being treated, for example, those with complementary activities
that do not adversely affect each other. In some embodiments, or in
addition, the composition can comprise an agent that enhances its
function, such as, for example, a cytotoxic agent, cytokine,
chemotherapeutic agent, or growth-inhibitory agent. Such molecules
are suitably present in combination in amounts that are effective
for the purpose intended.
[0361] In some embodiments, the dosage of the pharmaceutical
composition is a single dose or a repeated dose. In some
embodiments, the doses are given to a subject once per day, twice
per day, three times per day, or four or more times per day. In
some embodiments, about 1 or more (such as about 2 or more, about 3
or more, about 4 or more, about 5 or more, about 6 or more, or
about 7 or more) doses are given in a week. In some embodiments,
multiple doses are given over the course of days, weeks, months, or
years. In some embodiments, a course of treatment is about 1 or
more doses (such as about 2 or more does, about 3 or more doses,
about 4 or more doses, about 5 or more doses, about 7 or more
doses, about 10 or more doses, about 15 or more doses, about 25 or
more doses, about 40 or more doses, about 50 or more doses, or
about 100 or more doses).
[0362] In some embodiments, the pharmaceutical composition is
administered to a subject. Generally, dosages and routes of
administration of the pharmaceutical composition are determined
according to the size and condition of the subject, according to
standard pharmaceutical practice. For example, the therapeutically
effective dose can be estimated initially either in cell culture
assays or in animal models such as mice, rats, rabbits, dogs, pigs,
or monkeys. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans. The exact dosage will be determined
in light of factors related to the subject requiring treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active compound or to maintain the desired effect. Factors
that may be taken into account include the severity of the disease
state, the general health of the subject, the age, weight, and
gender of the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
The optimal dosage and treatment regime for a particular patient
can readily be determined by one skilled in the art of medicine by
monitoring the patient for signs of disease and adjusting the
treatment accordingly.
IV. METHODS OF USE AND THERAPEUTIC ADMINISTRATION
[0363] Also provided are methods for using and uses of the
multispecific polypeptide constructs. Such methods and uses include
therapeutic methods and uses, for example, involving administration
of the molecules or compositions containing the same, to a subject
having a disease, condition, or disorder, such as a tumor or
cancer. In some embodiments, the molecule and/or composition is
administered in an effective amount to effect treatment of the
disease or disorder. Uses include uses of the multispecific
polypeptide constructs in such methods and treatments, and in the
preparation of a medicament in order to carry out such therapeutic
methods. In some embodiments, the methods are carried out by
administering the multispecific polypeptide constructs, or
compositions comprising the same, to the subject having or
suspected of having the disease or condition. In some embodiments,
the methods thereby treat the disease or condition or disorder in
the subject.
[0364] In one embodiment, a multispecific polypeptide construct of
the disclosure may be used as therapeutic agents. Such agents will
generally be employed to diagnose, prognose, monitor, treat,
alleviate, and/or prevent a disease or pathology in a subject. A
therapeutic regimen is carried out by identifying a subject, e.g.,
a human patient or other mammal suffering from (or at risk of
developing) a disorder using standard methods. A multispecific
polypeptide construct is administered to the subject. A
multispecific polypeptide construct is administered to the subject
and will generally have an effect due to its binding with the
target(s).
[0365] In some embodiments, provided herein is a method of
modulating an immune response in a subject by administering a
therapeutically effective amount of any of the provided
multispecific conjugates or pharmaceutical compositions. In some
embodiments, the method of modulating an immune response increases
or enhances an immune response in a subject. For example, the
increase or enhanced response may be an increase in cell-mediated
immunity. In some examples, the method increases T-cell activity,
such as cytolytic T-cell (CTL) activity. In some embodiments, the
modulated (e.g., increased) immune response is against a tumor or
cancer.
[0366] Administration of the multispecific polypeptide construct
may activate innate immune cells via engagement of Fc.gamma.Rs
through the Fc-region of the multispecific polypeptide construct.
Administration of the multispecific polypeptide construct may
agonize, stimulate, activate, and/or augment innate immune cell
effector functions, including ADCC, cytokine release, degranulation
and/or ADCP. In examples where a multispecific polypeptide
construct contains a cleavable linker, administration of the
multispecific polypeptide construct may activate T-cell once the
linker(s) joining the first and second component is cleaved by a
protease thereby allowing the anti-CD3 binding portion to bind
CD3.epsilon. on the T cells. Administration of the multispecific
polypeptide construct may agonize, stimulate, activate, and/or
augment CD3-mediated T cell activation, cytotoxicity, cytokine
release and/or proliferation.
[0367] In some embodiments, the provided methods are for treating a
disease or condition in a subject by administering a
therapeutically effective amount of any of the provided
multispecific conjugates or pharmaceutical compositions. In some
embodiments, the disease or condition is a tumor or a cancer.
Generally, alleviation or treatment of a disease or disorder
involves the lessening of one or more symptoms or medical problems
associated with the disease or disorder. For example, in the case
of cancer, the therapeutically effective amount of the drug can
accomplish one or a combination of the following: reduce the number
of cancer cells; reduce the tumor size; inhibit (i.e., to decrease
to some extent and/or stop) cancer cell infiltration into
peripheral organs; inhibit tumor metastasis; inhibit, to some
extent, tumor growth; and/or relieve to some extent one or more of
the symptoms associated with the cancer. In some embodiments, a
composition of this disclosure can be used to prevent the onset or
reoccurrence of the disease or disorder in a subject, e.g., a human
or other mammal, such as a non-human primate, companion animal
(e.g., cat, dog, horse), farm animal, work animal, or zoo animal.
The terms subject and patient are used interchangeably herein.
[0368] In some embodiments, the pharmaceutical composition can be
used to inhibit growth of mammalian cancer cells (such as human
cancer cells). A method of treating cancer can include
administering an effective amount of any of the pharmaceutical
compositions described herein to a subject with cancer. The
effective amount of the pharmaceutical composition can be
administered to inhibit, halt, or reverse progression of cancers.
Human cancer cells can be treated in vivo, or ex vivo. In ex vivo
treatment of a human patient, tissue or fluids containing cancer
cells are treated outside the body and then the tissue or fluids
are reintroduced back into the patient. In some embodiments, the
cancer is treated in a human patient in vivo by administration of
the therapeutic composition into the patient.
[0369] Non-liming examples of disease include: all types of cancers
(breast, lung, colorectal, prostate, melanomas, head and neck,
pancreatic, etc.), rheumatoid arthritis, Crohn's disuse, SLE,
cardiovascular damage, ischemia, etc. For example, indications
would include leukemias, including T-cell acute lymphoblastic
leukemia (T-ALL), lymphoblastic diseases including multiple
myeloma, and solid tumors, including lung, colorectal, prostate,
pancreatic, and breast, including triple negative breast cancer.
For example, indications include bone disease or metastasis in
cancer, regardless of primary tumor origin; breast cancer,
including by way of non-limiting example, ER/PR+ breast cancer,
Her2+ breast cancer, triple-negative breast cancer; colorectal
cancer; endometrial cancer; gastric cancer; glioblastoma; head and
neck cancer, such as esophageal cancer; lung cancer, such as by way
of non-limiting example, non-small cell lung cancer; multiple
myeloma ovarian cancer; pancreatic cancer; prostate cancer;
sarcoma, such as osteosarcoma; renal cancer, such as by way of
nonlimiting example, renal cell carcinoma; and/or skin cancer, such
as by way of nonlimiting example, squamous cell cancer, basal cell
carcinoma, or melanoma. In some embodiments, the cancer is a
squamous cell cancer. In some embodiments, the cancer is a skin
squamous cell carcinoma. In some embodiments, the cancer is an
esophageal squamous cell carcinoma. In some embodiments, the cancer
is a head and neck squamous cell carcinoma. In some embodiments,
the cancer is a lung squamous cell carcinoma.
[0370] A therapeutically effective amount of a multispecific
polypeptide construct of the disclosure relates generally to the
amount needed to achieve a therapeutic objective. As noted above,
this may be a binding interaction between the multispecific
polypeptide construct and its target antigen(s) that, in certain
cases, agonize, stimulate, activate, and/or augment
Fc.gamma.R-mediated innate immune cell activation or CD3-mediated T
cell activation. The amount required to be administered will
furthermore depend on the binding affinity of the multispecific
polypeptide construct for its specific antigen(s), and will also
depend on the rate at which an administered multispecific
polypeptide construct is depleted from the free volume other
subject to which it is administered. Common ranges for
therapeutically effective dosing of a multispecific polypeptide
construct may be, by way of nonlimiting example, from about 0.01
.mu.g/kg body weight to about 10 mg/kg body weight. In some
embodiments, the therapeutically effective dosing of a
multispecific polypeptide construct of the disclosure may be, by
way of nonlimiting example, from about 0.01 mg/kg body weight to
about 5-10 mg/kg body weight. Common dosing frequencies may range,
for example, from twice daily to once a week.
[0371] Efficaciousness of treatment is determined in association
with any known method for diagnosing or treating the particular
disorder. Methods for the screening of multispecific polypeptide
construct that possess the desired specificity include, but are not
limited to, enzyme linked immunosorbent assay (ELISA) and other
immunologically mediated techniques known within the art. A variety
of means are known for determining if administration of the
provided multispecific polypeptide constructs sufficiently
modulates immunological activity by eliminating, sequestering, or
inactivating immune cells mediating or capable of mediating an
undesired immune response; inducing, generating, or turning on
immune cells that mediate or are capable of mediating a protective
immune response; changing the physical or functional properties of
immune cells; or a combination of these effects. Examples of
measurements of the modulation of immunological activity include,
but are not limited to, examination of the presence or absence of
immune cell populations (using flow cytometry,
immunohistochemistry, histology, electron microscopy, polymerase
chain reaction (PCR)); measurement of the functional capacity of
immune cells including ability or resistance to proliferate or
divide in response to a signal (such as using T-cell proliferation
assays and pepscan analysis based on 3H-thymidine incorporation
following stimulation with anti-CD3 antibody, anti-T-cell receptor
antibody, anti-CD28 antibody, calcium ionophores, PMA (phorbol
12-myristate 13-acetate) antigen presenting cells loaded with a
peptide or protein antigen; B cell proliferation assays);
measurement of the ability to kill or lyse other cells (such as
cytotoxic T cell assays); measurements of the cytokines,
chemokines, cell surface molecules, antibodies and other products
of the cells (e.g., by flow cytometry, enzyme-linked immunosorbent
assays, Western blot analysis, protein microarray analysis,
immunoprecipitation analysis); measurement of biochemical markers
of activation of immune cells or signaling pathways within immune
cells (e.g., Western blot and immunoprecipitation analysis of
tyrosine, serine or threonine phosphorylation, polypeptide
cleavage, and formation or dissociation of protein complexes;
protein array analysis; DNA transcriptional, profiling using DNA
arrays or subtractive hybridization); measurements of cell death by
apoptosis, necrosis, or other mechanisms (e.g., annexin V staining,
TUNEL assays, gel electrophoresis to measure DNA laddering,
histology; fluorogenic caspase assays, Western blot analysis of
caspase substrates); measurement of the genes, proteins, and other
molecules produced by immune cells (e.g., Northern blot analysis,
polymerase chain reaction, DNA microarrays, protein microarrays,
2-dimensional gel electrophoresis, Western blot analysis, enzyme
linked immunosorbent assays, flow cytometry); and measurement of
clinical symptoms or outcomes such as improvement of autoimmune,
neurodegenerative, and other diseases involving self-proteins or
self-polypeptides (clinical scores, requirements for use of
additional therapies, functional status, imaging studies) for
example, by measuring relapse rate or disease severity.
[0372] The multispecific polypeptide construct are also useful in a
variety of diagnostic and prophylactic formulations. In one
embodiment, a multispecific polypeptide construct is administered
to patients that are at risk of developing one or more of the
aforementioned disorders. A patient's or organ's predisposition to
one or more of the disorders can be determined using genotypic,
serological or biochemical markers.
[0373] In another embodiment of the disclosure, a multispecific
polypeptide construct is administered to human individuals
diagnosed with a clinical indication associated with one or more of
the aforementioned disorders. Upon diagnosis, a multispecific
polypeptide construct is administered to mitigate or reverse the
effects of the clinical indication.
[0374] Combination Therapies
[0375] In some embodiments, the multispecific polypeptide
constructs, conjugated multispecific polypeptide constructs, and
compositions thereof--referred to collectively herein as the
Therapeutic(s)--are administered in conjunction with one or more
additional agents, or a combination of additional agents. Suitable
additional agents include current pharmaceutical and/or surgical
therapies for an intended application. For example, the
Therapeutic(s) can be used in conjunction with an additional
chemotherapeutic or anti-neoplastic agent. For example, the
Therapeutic(s) and additional agent are formulated into a single
therapeutic composition, and the Therapeutic(s) and additional
agent are administered simultaneously. In some embodiments, the
Therapeutic(s) and additional agent are separate from each other,
e.g., each is formulated into a separate therapeutic composition,
and the Therapeutic(s) and the additional agent are administered
simultaneously, or the Therapeutic(s) and the additional agent are
administered at different times during a treatment regimen. For
example, the Therapeutic(s) is administered prior to the
administration of the additional agent, the Therapeutic(s) is
administered subsequent to the administration of the additional
agent, or the Therapeutic(s) and the additional agent are
administered in an alternating fashion. As described herein, the
Therapeutic(s) and additional agent are administered in single
doses or in multiple doses. In some embodiments, the additional
agent is coupled or otherwise attached to the Therapeutic(s).
Suitable additional agents are selected according to the purpose of
the intended application (i.e., killing, prevention of cell
proliferation, hormone therapy or gene therapy). Such agents may
include but is not limited to, for example, pharmaceutical agents,
toxins, fragments of toxins, alkylating agents, enzymes,
antibiotics, antimetabolites, antiproliferative agents, hormones,
neurotransmitters, DNA, RNA, siRNA, oligonucleotides, antisense
RNA, aptamers, diagnostics, radiopaque dyes, radioactive isotopes,
fluorogenic compounds, magnetic labels, nanoparticles, marker
compounds, lectins, compounds that alter cell membrane
permeability, photochemical compounds, small molecules, liposomes,
micelles, gene therapy vectors, viral vectors, and the like.
Finally, combinations of agents or combinations of different
classes of agents may be used.
[0376] In one embodiment, the multispecific polypeptide constructs
are administered in combination therapy, i.e., combined with other
agents, e.g., therapeutic agents, that are useful for treating
pathological conditions or disorders, such as autoimmune disorders
and inflammatory diseases. The term "in combination" in this
context means that the agents are given substantially
contemporaneously, either simultaneously or sequentially. If given
sequentially, at the onset of administration of the second
compound, the first of the two compounds is still detectable at
effective concentrations at the site of treatment.
[0377] For example, the combination therapy can include one or more
multispecific polypeptide constructs of the disclosure
co-formulated with, and/or co-administered with, one or more
additional therapeutic agents, e.g., one or more cytokine and
growth factor inhibitors, immunosuppressants, anti-inflammatory
agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic
or cytostatic agents, as described in more detail below.
Furthermore, one or more multispecific polypeptide constructs
described herein may be used in combination with two or more of the
therapeutic agents described herein. Such combination therapies may
advantageously utilize lower dosages of the administered
therapeutic agents, thus avoiding possible toxicities or
complications associated with the various monotherapies.
[0378] In other embodiments, one or more multispecific polypeptide
constructs of the disclosure can be co-formulated with, and/or
co-administered with, one or more anti-inflammatory drugs,
immunosuppressants, or metabolic or enzymatic inhibitors.
Nonlimiting examples of the drugs or inhibitors that can be used in
combination with the antibodies described herein, include, but are
not limited to, one or more of: nonsteroidal anti-inflammatory
drug(s) (NSAIDs), e.g., ibuprofen, tenidap, naproxen, meloxicam,
piroxicam, diclofenac, and indomethacin; sulfasalazine;
corticosteroids such as prednisolone; cytokine suppressive
anti-inflammatory drug(s) (CSAIDs); inhibitors of nucleotide
biosynthesis, e.g., inhibitors of purine biosynthesis, folate
antagonists (e.g., methotrexate
(N-[4-[[(2,4-diamino-6-pteridinyl)methyl] methylamino]
benzoyl]-L-glutamic acid); and inhibitors of pyrimidine
biosynthesis, e.g., dihydroorotate dehydrogenase (DHODH)
inhibitors. Suitable therapeutic agents for use in combination with
the antibodies of the disclosure include NSAIDs, CSAIDs, (DHODH)
inhibitors (e.g., leflunomide), and folate antagonists (e.g.,
methotrexate).
[0379] Examples of additional inhibitors include one or more of:
corticosteroids (oral, inhaled and local injection);
immunosuppressants, e.g., cyclosporin, tacrolimus (FK-506); and
mTOR inhibitors, e.g., sirolimus (rapamycin--RAPAMUNE.TM. or
rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g.,
ester rapamycin derivatives, e.g., CCI-779); agents that interfere
with signaling by proinflammatory cytokines such as TNF.alpha. or
IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors); COX2
inhibitors, e.g., celecoxib, rofecoxib, and variants thereof;
phosphodiesterase inhibitors, e.g., R973401 (phosphodiesterase Type
IV inhibitor); phospholipase inhibitors, e.g., inhibitors of
cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketone
analogs); inhibitors of vascular endothelial cell growth factor or
growth factor receptor, e.g., VEGF inhibitor and/or VEGF-R
inhibitor; and inhibitors of angiogenesis. Suitable therapeutic
agents for use in combination with the antibodies of the disclosure
are immunosuppressants, e.g., cyclosporin, tacrolimus (FK-506);
mTOR inhibitors, e.g., sirolimus (rapamycin) or rapamycin
derivatives, e.g., soluble rapamycin derivatives (e.g., ester
rapamycin derivatives, e.g., CCI-779); COX2 inhibitors, e.g.,
celecoxib and variants thereof; and phospholipase inhibitors, e.g.,
inhibitors of cytosolic phospholipase 2 (cPLA2), e.g.,
trifluoromethyl ketone analogs. Additional examples of therapeutic
agents that can be combined with a multispecific polypeptide
construct include one or more of: 6-mercaptopurines (6-MP);
azathioprine sulphasalazine; mesalazine; olsalazine;
chloroquine/hydroxychloroquine (PLAQUENIL.RTM.); pencillamine;
aurothiornalate (intramuscular and oral); azathioprine; coichicine;
beta-2 adrenoreceptor agonists (salbutamol, terbutaline,
salmeteral); xanthines (theophylline, aminophylline); cromoglycate;
nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate
mofetil; adenosine agonists; antithrombotic agents; complement
inhibitors; and adrenergic agents.
V. EXEMPLARY EMBODIMENTS
[0380] Among the provided embodiments are:
[0381] 1. A multispecific polypeptide construct, the multispecific
polypeptide construct comprising a first component comprising an
immunoglobulin Fc region and a second component comprising a
CD3-binding region, wherein:
[0382] the first and second components are coupled by a
non-cleavable linker, wherein the Fc region is positioned
N-terminal to the CD3-binding region; and
[0383] one or both of the first and second components comprises an
antigen binding domain that binds a tumor associated antigen
(TAA).
[0384] 2. The multispecific polypeptide construct of embodiment 1,
wherein the CD3-binding region binds CD3 (CD3.epsilon.).
[0385] 3. The multispecific construct of embodiment 1 or embodiment
2, wherein the antigen binding domain is positioned
amino-terminally relative to the Fc region and/or
carboxy-terminally relative to the CD3 binding region of the
multispecific polypeptide construct.
[0386] 4. The multispecific polypeptide construct of any of
embodiments 1-3, wherein the first component comprises a first
antigen binding domain and the second component comprises a second
antigen binding domain, wherein each of the antigen binding domains
bind a tumor associated antigen (TAA).
[0387] 5. The multispecific polypeptide construct of embodiment 4,
wherein the first antigen binding domain is positioned
amino-terminally relative to the Fc region of the multispecific
construct and the second antigen binding domain is positioned
carboxy-terminally relative to the CD3 binding region of the
multispecific construct.
[0388] 6. A multispecific polypeptide construct, wherein the
multispecific construct comprises in order, from N-terminus to
C-terminus:
[0389] a first antigen binding domain that binds to a
tumor-associated antigen (TAA);
[0390] an immunoglobulin Fc region;
[0391] a non-cleavable linker;
[0392] a CD3 binding region that binds CD3 (CD3.epsilon.); and
[0393] a second antigen binding domain that binds a
tumor-associated antigen (TAA).
[0394] 7. A multispecific polypeptide construct, wherein the
multispecific construct comprises in order, from N-terminus to
C-terminus:
[0395] an immunoglobulin Fc region;
[0396] a non-cleavable linker;
[0397] a CD3 binding region that binds CD3 (CD3.epsilon.); and
[0398] an antigen binding domain that binds a tumor-associated
antigen (TAA).
[0399] 8. A multispecific polypeptide construct, wherein the
multispecific construct comprises in order, from N-terminus to
C-terminus:
[0400] an antigen binding domain that binds to a tumor-associated
antigen (TAA);
[0401] an immunoglobulin Fc region;
[0402] a non-cleavable linker; and
[0403] a CD3 binding region that binds CD3 (CD3.epsilon.).
[0404] 9. The multispecific polypeptide construct of any of
embodiments 1-8, wherein the Fc region is a homodimeric Fc
region.
[0405] 10. The multispecific polypeptide construct of any of
embodiments 1-9, wherein the Fc region is an Fc region of a human
IgG1, a human IgG2, a human IgG3, or a human IgG4, or is an
immunologically active fragment thereof.
[0406] 11. The multispecific polypeptide construct of any of
embodiments 1-10, wherein the Fc region comprises a polypeptide
comprises the amino acid sequence set forth in SEQ ID NO: 1 or a
sequence of amino acids that has at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:1.
[0407] 12. The multispecific polypeptide construct of any of
embodiments 1-10, wherein the Fc region comprises a polypeptide
comprises the amino acid sequence set forth in SEQ ID NO: 2 or a
sequence of amino acids that has at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2;
[0408] the Fc region comprises a polypeptide comprises the amino
acid sequence set forth in SEQ ID NO: 4 or a sequence of amino
acids that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% sequence identity to SEQ ID NO:4; or
[0409] the Fc region comprises a polypeptide comprises the amino
acid sequence set forth in SEQ ID NO: 5 or a sequence of amino
acids that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% sequence identity to SEQ ID NO:5.
[0410] 13. The multispecific polypeptide construct of any of
embodiments 1-6, 9 and 12, wherein the Fc region is a heterodimeric
Fc region.
[0411] 14. The multispecific polypeptide construct of embodiment
13, wherein one or both Fc polypeptides of the heterodimeric Fc
region comprises at least one modification to induce
heterodimerization compared to a polypeptide of a homodimeric Fc
region, optionally compared to the Fc polypeptide set forth in SEQ
ID NO:1 or an immunologically active fragment thereof.
[0412] 15. The multispecific polypeptide construct of embodiment
14, wherein each of the Fc polypeptides of the heterodimeric Fc
independently comprise at least one amino acid modification.
[0413] 16. The multispecific polypeptide construct of embodiment
15, wherein each of the Fc polypeptides of the heterodimeric Fc
comprise a knob-into-hole modification or comprise a charge
mutation to increase electrostatic complementarity of the
polypeptides.
[0414] 17. The multispecific polypeptide construct of embodiment
16, wherein the amino acid modification is a knob-into-hole
modification.
[0415] 18. The multispecific polypeptide construct of any of
embodiments 13-17, wherein the first Fc polypeptide of the
heterodimeric Fc comprises the modification selected from among
Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof and the
second Fc polypeptide of the heterodimeric Fc comprises the
modification T366W.
[0416] 19. The multispecific polypeptide construct of embodiment
18, wherein the first and second Fc polypeptides further comprises
a modification of a non-cysteine residue to a cysteine residue,
wherein the modification of the first polypeptide is at one of the
position Ser354 and Y349 and the modification of the second Fc
polypeptide is at the other of the position Ser354 and Y349.
[0417] 20. The multispecific polypeptide construct of embodiment
16, wherein the amino acid modification is a charge mutation to
increase electrostatic complementarity of the polypeptides.
[0418] 21. The multispecific polypeptide construct of any of
embodiments 13-16 and 20, wherein the first and/or second Fc
polypeptides or each of the first and second Fc polypeptide
comprise a modification in complementary positions, wherein the
modification is replacement with an amino acid having an opposite
charge to the complementary amino acid of the other
polypeptide.
[0419] 22. The multispecific polypeptide construct of any of
embodiments 14-21, wherein one of the first or second Fc
polypeptide of the heterodimeric Fc further comprises a
modification at residue Ile253.
[0420] 23. The multispecific polypeptide construct of embodiment
22, wherein the modification is Ile253Arg.
[0421] 24. The multispecific polypeptide construct of any of
embodiments 14-23, wherein one of the first or second Fc
polypeptide of the heterodimeric Fc further comprises a
modification at residue His435.
[0422] 25. The multispecific polypeptide construct of embodiment
24, wherein the modification is His435Arg.
[0423] 26. The multispecific polypeptide construct of any of
embodiments 1-25, wherein the Fc region comprises a polypeptide
that lacks Lys447.
[0424] 27. The multispecific polypeptide construct of any of
embodiments 1-26, wherein the Fc region comprises a polypeptide
comprising at least one modification to enhance FcRn binding.
[0425] 28. The multispecific polypeptide construct of embodiment
27, wherein the modification is at a position selected from the
group consisting of Met252, Ser254, Thr256, Met428, Asn434, and
combinations thereof.
[0426] 29. The multispecific polypeptide construct of embodiment
28, wherein the modification is at a position selected from the
group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V,
Asn434S, and combinations thereof.
[0427] 30. The multispecific polypeptide construct of embodiment
28, wherein the modification is at position Met252 and at position
Met428.
[0428] 31. The multispecific polypeptide construct of embodiment
30, wherein the modification is Met252Y and Met428L.
[0429] 32. The multispecific polypeptide construct of embodiment
30, wherein the modification is Met252Y and Met428V.
[0430] 33. The multispecific polypeptide construct of any of
embodiments 13-32, wherein the first polypeptide of the
heterodimeric Fc comprises the sequence of amino acids set forth in
any of SEQ ID NOS:82, 86, 94 or 96, and the second polypeptide of
the heterodimeric Fc comprises the sequence of amino acids set
forth in any of SEQ ID NOS:83, 87, 90, 92, 98 or 100.
[0431] 34. The multispecific polypeptide construct of any of
embodiments 1-33, wherein the Fc region comprises a polypeptide
comprising at least one amino acid modification that reduces
effector function and/or reduces binding to an effector molecule
selected from an Fc gamma receptor or C1q.
[0432] 35. The multispecific polypeptide construct of embodiment
34, wherein the one or more amino acid modification is deletion of
one or more of Glu233, Leu234 or Leu235.
[0433] 36. The multispecific polypeptide construct of any of
embodiments 13-32, 34 and 35, wherein the first polypeptide of the
heterodimeric Fc comprises the sequence of amino acids set forth in
any of SEQ ID NOS: 84, 88, 95 or 97 and the second polypeptide of
the heterodimeric Fc comprises the sequence of amino acids set
forth in any of SEQ ID NOS: 85, 89, 91, 93, 99 or 101.
[0434] 37. The multispecific polypeptide construct of any of
embodiments 1-32, wherein the Fc region comprises a polypeptide
comprising at least one modification to enhance Fc.gamma.R
binding.
[0435] 38. The multispecific polypeptide construct of embodiment
37, wherein the modification is modification at Ser239 or
Ile332.
[0436] 39. The multispecific polypeptide construct of any of
embodiments 1-32 and 37, wherein the glycosylation of the Fc region
is modified to enhance Fc.quadrature.R binding as compared to an
unmodified Fc region.
[0437] 40. The multispecific polypeptide construct of embodiment
39, wherein the Fc region lacks or has reduced fucose content.
[0438] 41. The multispecific polypeptide construct of any of
embodiments 1-40, wherein the CD3 binding region is an anti-CD3
antibody or antigen-binding fragment.
[0439] 42. The multispecific polypeptide construct of embodiment
41, wherein the anti-CD3 antibody or antigen binding fragment
comprises a variable heavy chain region (VH) and a variable light
chain region (VL).
[0440] 43. The multispecific polypeptide construct of any of
embodiments 1-42, wherein the CD3 binding region is monovalent.
[0441] 44. The multispecific polypeptide construct of any of
embodiments 41-43, wherein the anti-CD3 antibody or antigen binding
fragment is not a single chain antibody, optionally is not a single
chain variable fragment (scFv).
[0442] 45. The multispecific polypeptide construct of embodiment 42
or embodiment 44, wherein the Fc is a heterodimeric Fc and the VH
and VL that comprise the anti-CD3 antibody or antigen binding
fragment are linked to opposite polypeptides of the heterodimeric
Fc.
[0443] 46. The multispecific polypeptide construct of any of
embodiments 1-45, wherein the CD3 binding region is not able to, or
is not substantially able to, bind or engage CD3 unless at least
one of the antigen binding domain is bound to its TAA.
[0444] 47. The multispecific polypeptide construct of any of
embodiments 1-46, wherein the CD3 binding region is not able to, or
is not substantially able, to bind or engage CD3 unless at least
two of the antigen binding domain is bound to its TAA.
[0445] 48. The multispecific polypeptide construct of any of
embodiments 1-47, wherein the linker is a polypeptide linker.
[0446] 49. The multispecific polypeptide construct of embodiment
48, wherein the linker is a polypeptide of up to 25 amino acids in
length.
[0447] 50. The multispecific polypeptide construct of embodiment 48
or embodiment 49, wherein the linker is a polypeptide of from or
from about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino
acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino
acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids,
6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to
12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24
amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14
amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24
amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14
amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20
amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24
amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24
amino acids, 18 to 20 amino acids or 20 to 24 amino acids.
[0448] 51. The multispecific polypeptide construct of any of
embodiments 48-50, wherein the linker is a polypeptide that is 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
amino acids in length.
[0449] 52. The multispecific polypeptide construct of any of
embodiments 48-51, wherein the linker is a polypeptide that is 3 to
18 amino acids in length.
[0450] 53. The multispecific polypeptide construct of any of
embodiments 48-51, wherein the linker is a polypeptide that is 12
to 18 amino acids in length.
[0451] 54. The multispecific polypeptide construction of any of
embodiments 48-51, wherein the linker is a polypeptide that is 15
to 18 amino acids in length.
[0452] 55. A multispecific polypeptide construct, the multispecific
polypeptide construct comprising a first component comprising a
heterodimeric Fc region and a second component comprising an
anti-CD3 antibody or antigen-binding fragment comprising a variable
heavy chain region (VH) and a variable light chain region (VL),
wherein:
[0453] the VH and VL that comprise the anti-CD3 antibody or antigen
binding fragment are linked to opposite polypeptides of the
heterodimeric Fc;
[0454] the first and second components are coupled by a
non-cleavable linker, wherein the heterodimeric Fc region is
positioned N-terminal to the anti-CD3 antibody; and
[0455] one or both of the first and second components comprises an
antigen binding domain that binds a tumor associated antigen
(TAA).
[0456] 56. The multispecific polypeptide construct of any of
embodiments 52-55, wherein the non-cleavable linker does not
contain a substrate recognition site that is specifically
recognized for cleavage by a protease.
[0457] 57. The multispecific polypeptide construct of embodiment
56, wherein the protease is produced by an immune effector cell, by
a tumor, or by cells present in the tumor microenvironment.
[0458] 58. The multispecific polypeptide construct of embodiment
57, wherein the protease is produced by an immune effector cell and
the immune effector cell is an activated T cell, a natural killer
(NK) cell, or an NK T cell.
[0459] 59. The multispecific polypeptide construct of any of
embodiments 56-58, wherein the protease is selected from among
matriptase, a matrix metalloprotease (MMP), Granzyme B, and
combinations thereof.
[0460] 60. The multispecific polypeptide construct of embodiment
59, wherein the protease is granzyme B.
[0461] 61. The multispecific polypeptide construct of any of
embodiments 1-60, wherein the non-cleavable linker comprises GS,
GGS, GGGGS (SEQ ID NO:149), GGGGGS (SEQ ID NO:135) and combinations
thereof.
[0462] 62. The multispecific polypeptide construct of any of
embodiments 1-61, wherein the non-cleavable linker comprises
(GGS)n, wherein n is 1 to 10.
[0463] 63. The multispecific polypeptide construct of any of
embodiments 1-62, wherein the non-cleavable linker comprises
(GGGGS)n (SEQ ID NO: 173), wherein n is 1 to 10.
[0464] 64. The multispecific polypeptide construct of any of
embodiments 1-62, wherein the non-cleavable linker comprises
(GGGGGS)n (SEQ ID NO:172), wherein n is 1 to 4.
[0465] 65. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises
GGS.
[0466] 66. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises GGGGS
(SEQ ID NO: 149).
[0467] 67. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises GGGGGS
(SEQ ID NO: 135).
[0468] 68. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises (GGS)2
(SEQ ID NO: 10).
[0469] 69. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises
GGSGGSGGS (SEQ ID NO: 11).
[0470] 70. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises
GGSGGSGGSGGS (SEQ ID NO: 12).
[0471] 71. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises
GGSGGSGGSGGSGGS (SEQ ID NO: 13).
[0472] 72. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises
GGGGGSGGGGGSGGGGGS (SEQ ID NO: 119).
[0473] 73. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises
GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147)
[0474] 74. The multispecific polypeptide construct of any of
embodiments 1-64, wherein the non-cleavable linker comprises and
GGGGSGGGGSGGGGS (SEQ ID NO:170).
[0475] 75. The multispecific polypeptide construct of any of
embodiments 45-74, wherein the multispecific polypeptide construct
comprises at least (i) a first polypeptide comprising the first Fc
polypeptide of the heterodimeric Fc region, the linker and the VH
domain of the anti-CD3 antibody or antigen binding fragment; and
(ii) a second polypeptide comprising the second Fc polypeptide of
the heterodimeric Fc region, the linker and the VL domain of the
anti-CD3 antibody or antigen binding fragment, wherein one or both
of the first and second polypeptide comprise at least one
antigen-binding domain that binds to a tumor associated antigen
(TAA).
[0476] 76. The multispecific polypeptide construct of any of
embodiments 1-75, wherein one or more antigen binding domain that
binds TAA results in monovalent, bivalent, trivalent, or
tetravalent binding to the TAA.
[0477] 77. The multispecific polypeptide construct of embodiment
75, wherein only one of the first or second polypeptide comprises
the at least one antigen-binding domain that binds a TAA.
[0478] 78. The multispecific polypeptide construct of embodiment 75
or embodiment 77, wherein the at least one antigen binding domain
is positioned amino-terminally relative to the Fc region and/or is
positioned carboxy-terminally relative to the CD3 binding region of
one of the first or second polypeptide of the multispecific
polypeptide construct.
[0479] 79. The multispecific polypeptide construct of embodiment 75
or embodiment 77, wherein the at least one antigen binding domain
is positioned amino-terminally relative to the Fc region of the
multispecific construct and the second antigen binding domain is
positioned carboxy-terminally relative to the CD3 binding region of
the multispecific construct.
[0480] 80. The multispecific polypeptide construct of any of
embodiments 1-79, wherein the antigen binding domain, or
independently each of the antigen binding domains, comprises an
extracellular domain or binding fragment thereof of the native
cognate binding partner of the TAA, or a variant thereof that
exhibits binding activity to the TAA.
[0481] 81. The multispecific polypeptide construct of any of
embodiments 1-79, wherein the antigen binding domain, or
independently each of the antigen binding domains, is an antibody
or antigen-binding fragment thereof selected from the group
consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a
scFv, a scAb, a dAb, a single domain heavy chain antibody, and a
single domain light chain antibody.
[0482] 82. The multispecific polypeptide construct of embodiment
81, wherein the antibody or antigen-binding fragment thereof is a
Fv, a scFv, a Fab, a single domain antibody (sdAb), a VNAR, or a
VHH.
[0483] 83. The multispecific polypeptide construct of embodiment 81
or embodiment 82, wherein the antibody or antigen-binding fragment
is an sdAb.
[0484] 84. The multispecific polypeptide construct of embodiment
83, wherein the sdAb is a human or humanized sdAb.
[0485] 85. The multispecific polypeptide construct of embodiment 83
or embodiment 84, wherein the sdAb is VHH, VNAR, an engineered VH
domain or an engineered VK domain.
[0486] 86. The multispecific polypeptide construct of embodiment 81
or embodiment 82, wherein the antibody or antigen-binding fragment
thereof is an scFv.
[0487] 87. The multispecific polypeptide construct of embodiment 81
or embodiment 82, wherein the antibody or antigen-binding fragment
thereof is a Fab.
[0488] 88. The multispecific polypeptide construct of embodiment
87, wherein the multispecific polypeptide construct comprises:
[0489] (i) a first polypeptide comprising the first Fc polypeptide
of the heterodimeric Fc region, the linker and the VH domain of the
anti-CD3 antibody or antigen binding fragment;
[0490] (ii) a second polypeptide comprising the second Fc
polypeptide of the heterodimeric Fc region, the linker and the VL
domain of the anti-CD3 antibody or antigen binding fragment,
and
[0491] (iii) a third polypeptide comprising a VH-CH1 (Fd) or VL-CL
of a Fab antibody fragment that binds to a tumor-associated
antigen, wherein the first and/or second polypeptide further
comprises the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody
fragment.
[0492] 89. The multispecific polypeptide construct of embodiment
88, wherein only one of the first or second polypeptide comprises
the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody
fragment.
[0493] 90. The multispecific polypeptide construct of embodiment
89, wherein both the first or second polypeptide comprises the
other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment.
[0494] 91. The multispecific polypeptide construct of embodiment 89
or embodiment 90, wherein the other of the VH-CH1 (Fd) or VL-CL of
the Fab antibody fragment is positioned amino-terminally relative
to the Fc region and/or at the carboxy-terminally relative to the
CD3 binding region of one of the first or second polypeptide of the
multispecific polypeptide construct.
[0495] 92. The multispecific polypeptide construct of any of
embodiments 89-91, wherein the other of the VH-CH1 (Fd) or VL-CL of
the Fab antibody fragment is positioned amino-terminally relative
to the Fc region of the first polypeptide or second polypeptide and
at the carboxy-terminally relative to the CD3 binding region of the
other of the first or second polypeptide.
[0496] 93. The multispecific polypeptide construct of any of
embodiments 1-92, wherein the antigen binding domain, or
independently each of the antigen binding domains, binds to a tumor
antigen selected from among 1-92-LFA-3, 5T4, Alpha-4 integrin,
Alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2,
Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA,
C5 complement, C-242, CA9, CA19-9, (Lewis a), Carbonic anhydrase 9,
CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27,
CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47,
CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95,
CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166,
CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3,
CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF,
CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4,
DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin B receptor
(ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP,
FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor
alpha (FR.alpha.), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4,
GM-CSF, GM-CSFR, GP IIb/IIIa receptors, Gp130, GPIIB/IIIA, GPNMB,
GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS,
IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R,
IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbeta1, IL13,
IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27/IL27R (wsx1),
IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin
Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,
LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP,
Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF,
Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4,
NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta,
PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR,
RAAG12, RAGE, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2,
TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6,
TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFRS12A, TRAIL-R1,
TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR,
VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2,
VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.
[0497] 94. The multispecific polypeptide construct of any of
embodiments 1-93, wherein multispecific antigen binding domain
comprises at least a first antigen binding domain and a second
antigen binding domain, wherein the first antigen binding domain
and second antigen binding domain bind to the same TAA.
[0498] 95. The multispecific polypeptide construct of embodiment
94, wherein the first antigen binding domain and the second antigen
binding domain binds a different epitope of the same TAA.
[0499] 96. The multispecific polypeptide construct of embodiment
94, wherein the first antigen binding domain and the second antigen
binding domain binds the same epitope of the same TAA.
[0500] 97. The multispecific polypeptide construct of any of
embodiments 1-96, wherein multispecific antigen binding domain
comprises at least a first antigen binding domain and a second
antigen binding domain wherein the first antigen binding domain and
the second antigen binding domain bind a different TAA.
[0501] 98. The multispecific polypeptide construct of any of
embodiments 5-97, wherein the multispecific polypeptide construct
comprises a first linking peptide (LP1) between the first antigen
binding domain and the Fc region.
[0502] 99. The multispecific polypeptide construct of any of
embodiments 5-98, wherein the multispecific polypeptide construct
comprises a second linking peptide (LP2) between the CD3 binding
region and the second antigen binding domain.
[0503] 100. The multispecific polypeptide construct of any of
embodiments 5-99, wherein the multispecific polypeptide construct
comprises a first linking peptide (LP1) between the first antigen
binding domain and the Fc region and a second linking peptide (LP2)
between the CD3 binding region and the second antigen binding
domain, and wherein the multispecific polypeptide construct has the
structural arrangement from N-terminus to C-terminus as follows:
first antigen binding domain-LP1-Fc region-non-cleavable linker-CD3
binding region-LP2-second antigen binding domain.
[0504] 101. The multispecific polypeptide construct of embodiment
100, wherein the two linking peptides are identical to each
other.
[0505] 102. The multispecific polypeptide construct of embodiment
100, wherein the two linking peptides are not identical to each
other.
[0506] 103. The multispecific polypeptide construct of any of
embodiments 98-102, wherein LP1 or LP2 is independently a peptide
of about 1 to 20 amino acids in length.
[0507] 104. The multispecific polypeptide of embodiment 103,
wherein LP1 or LP2 independently comprise a peptide that is or
comprises any Gly-Ser linker as set forth in SEQ ID NOs: 10-13,
119, 135, 147, 149 or GGS.
[0508] 105. The multispecific polypeptide construct of any of
embodiments 41-104, wherein the anti-CD3 antibody or antigen
binding fragment is an Fv antibody fragment.
[0509] 106. The multispecific polypeptide construct of embodiment
105, wherein the Fv antibody fragment comprises a disulfide
stabilized anti-CD3 binding Fv fragment (dsFv).
[0510] 107. The multispecific polypeptide construct of any of
embodiments 41-106, wherein:
[0511] the anti-CD3 antibody or antigen-binding fragment comprises
a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 16)
or SEQ ID NO:211; a VH CD2 comprising the amino acid sequence
RIRSKYNNYATYYADSVKD (SEQ ID NO: 17) or SEQ ID NO:212; a VH CDR3
comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18),
a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID
NO: 19); a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ
ID NO: 20); and a VL CDR3 comprising the amino acid sequence
ALWYSNLWV (SEQ ID NO: 21): or the anti-CD3 antibody or
antigen-binding fragment comprises a VH CDR1 sequence that includes
at least the amino acid sequence GFTFNTYAMN (SEQ ID NO: 211); a VH
CDR2 sequence that includes at least the amino acid sequence
RIRSKYNNYATY (SEQ ID NO: 212); a VH CDR3 sequence that includes at
least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL
CDR1 sequence that includes at least the amino acid sequence
GSSTGAVTTSNYAN (SEQ ID NO: 229); a VL CDR2 sequence that includes
at least the amino acid sequence GTNKRAP (SEQ ID NO: 230); and a VL
CDR3 sequence that includes at least the amino acid sequence
ALWYSNHWV (SEQ ID NO: 225).
[0512] 108. The multispecific polypeptide construct of embodiment
106 or embodiment 107, wherein the anti-CD3 dsFv comprises:
[0513] a VH having the amino acid sequence of any of SEQ ID NOS: 14
and 32-62 or a sequence that exhibits at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID
NOS: 14 and 32-62; and
[0514] a VL having the amino acid sequence of any of SEQ ID NOS: 15
and 63-81 or a sequence that exhibits at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID
NOS: 14 and 32-62.
[0515] 109. The multispecific polypeptide construct of any of
embodiments 106-108, wherein the anti-CD3 dsFv comprises the amino
acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ
ID NO: 15.
[0516] 110. The multispecific polypeptide construct of any of
embodiments 102-104, wherein the anti-CD3 dsFv comprises the amino
acid sequence of SEQ ID NO: 44 and the amino acid sequence of SEQ
ID NO: 72.
[0517] 111. The multispecific polypeptide construct of any of
embodiments 1-109, wherein the multispecific polypeptide construct
is conjugated to an agent.
[0518] 112. The multispecific polypeptide construct of embodiment
111, wherein the agent is a therapeutic agent, an antineoplastic
agent, a toxin or fragment thereof, a detectable moiety or a
diagnostic agent.
[0519] 113. The multispecific polypeptide construct of embodiment
112, wherein the agent is conjugated to the multispecific
polypeptide construct via a linker.
[0520] 114. A polynucleotide(s) encoding the mutispecific
polypeptide constructs of any of embodiments 1-113.
[0521] 115. A polynucleotide encoding a polypeptide chain of any of
the mutispecific polypeptide constructs of any of embodiments
1-113.
[0522] 116. A polynucleotide, comprising a first nucleic acid
sequence encoding a first polypeptide of a mutispecific construct
of any of embodiments 1-115 and a second nucleic acid sequence
encoding a second polypeptide of the mutispecific construct,
wherein the first and second nucleic acid sequence are separated by
an internal ribosome entry site (IRES), or a nucleic acid encoding
a self-cleaving peptide or a peptide that causes ribosome
skipping.
[0523] 117. The polynucleotide of embodiment 116, wherein the first
nucleic acid sequence and second nucleic acid sequence are operably
linked to the same promoter.
[0524] 118. The polynucleotide of embodiment 116 or embodiment 117,
wherein the multispecific polypeptide construct comprises a third
polypeptide chain, and the polynucleotide further comprises a third
nucleic acid encoding the third polypeptide of the mutispecific
construct.
[0525] 119. The polynucleotide of embodiment 118, wherein the third
nucleic acid is separated from the first and/or second polypeptide
by an internal ribosome entry site (IRES), or a nucleic acid
encoding a self-cleaving peptide or a peptide that causes ribosome
skipping and/or the third nucleic acid sequence is operably linked
to the same promoter as the first and/or second nucleic acid
sequence.
[0526] 120. The polynucleotide of any of embodiments 116-119,
wherein the nucleic acid encoding a self-cleaving peptide or a
peptide that causes ribosome skipping is selected from a T2A, a
P2A, a E2A or a F2A.
[0527] 121. A vector, comprising the polynucleotide of any of
embodiments 114-120.
[0528] 122. The vector of embodiment 121 that is an expression
vector.
[0529] 123. The vector of embodiment 121 or 122 that is a viral
vector or a eukaryotic vector, optionally wherein the eukaryotic
vector is a mammalian vector.
[0530] 124. A cell, comprising polynucleotide or polynucleotides of
any of embodiments 114-120, or a vector or vectors of any of
embodiments 121-123.
[0531] 125. The cell of embodiment 124, wherein the cell is
recombinant or isolated.
[0532] 126. The cell of embodiment 125, wherein the cell is a
mammalian cell.
[0533] 127. The cell of embodiment 126, wherein the cell is a
HEK293 or CHO cell.
[0534] 128. A method of producing a multispecific polypeptide
construct, the method comprising introducing into a cell a
polynucleotide or polynucleotides of any of embodiments 114-120 or
a vector or vectors of any of embodiments 121-123 and culturing the
cell under conditions to produce the multispecific polypeptide
construct.
[0535] 129. A method of producing a multispecific polypeptide
construct, the method comprising culturing the cell of any of
embodiments 124-127 under conditions in which the multispecific
polypeptide is produced by the cell.
[0536] 130. The cell of embodiment 128 or 129, wherein the cell is
a mammalian cell.
[0537] 131. The cell of embodiment 130, wherein the cell is a
HEK293 or CHO cell.
[0538] 132. The method of embodiment 128 or embodiment 129, further
comprising isolating or purifying the multispecific polypeptide
construct from the cell.
[0539] 133. The method of any of embodiments 128-132, wherein the
multispecific polypeptide construct is a heterodimer.
[0540] 134. A multispecific polypeptide construct produced by the
method of any of embodiments 128-133.
[0541] 135. A pharmaceutical composition comprising the
multispecific polypeptide construct of any of embodiments 1-113 or
embodiment 134 and a pharmaceutically acceptable carrier.
[0542] 136. The pharmaceutical composition of embodiment 135 that
is sterile.
[0543] 137. A method of stimulating or inducing an immune response,
the method comprising contacting a target cell and a T cell with
the multispecific polypeptide construct of any of embodiments 1-113
or embodiment 134 or the pharmaceutical composition of embodiments
109 or embodiment 110, said target cell expressing a tumor
associated antigen recognized by the multispecific polypeptide
construct.
[0544] 138. The method of embodiment 137, wherein the target cell
is a tumor cell expressing the tumor associated antigen (TAA).
[0545] 139. The method of embodiment 137 or embodiment 138, wherein
the contacting is carried out ex vivo or in vitro.
[0546] 140. The method of any of embodiments 137-149, wherein the
contacting is carried out in vivo in a subject.
[0547] 141. A method of stimulating or inducing an immune response
in a subject, the method comprising administering, to a subject in
need thereof, a therapeutically effective amount of the
multispecific conjugate of any of embodiments 1-113 or embodiment
134 or the pharmaceutical composition of embodiments 109 or
embodiment 110.
[0548] 142. The method of any of embodiments 137-141, which
increases cell-mediated immunity.
[0549] 143. The method of any of embodiments 137-142, which
increases T-cell activity.
[0550] 144. The method of any of embodiments 137-143, which
increases cytolytic T-cell (CTL) activity.
[0551] 145. The method of any of embodiments 137-144, wherein the
immune response is increased against a tumor or cancer.
[0552] 146. The method of any of embodiments 137-145, wherein the
method treats a disease or condition in the subject.
[0553] 147. A method of treating a disease or condition in a
subject, the method comprising administering, to a subject in need
thereof, a therapeutically effective amount of the multispecific
conjugate of any of embodiments 1-113 or the pharmaceutical
composition of embodiments 135 or embodiment 136.
[0554] 148. The method of embodiment 146 or embodiment 147, wherein
the disease or condition is a tumor or a cancer.
[0555] 149. The method of any of embodiments 140-148, wherein said
subject is a human.
VI. EXAMPLES
[0556] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1: Method of Producing Multispecific Constructs With
Constrained CD3 Binding
[0557] Example 1 describes the generation and expression of
multispecific polypeptide constructs containing a CD3 binding
region that exhibits constrained CD3 binding. The multispecific
constructs were generated in various configurations, as shown in
FIG. 1, FIGS. 2A-2B, FIGS. 3A-3C and FIGS. 4A-4B, to contain a
heterodimeric Fc region of an immunoglobulin coupled by a linker
(e.g. a non-cleavable linker) to the CD3 binding region, and one or
more antigen binding domains that binds a tumor associated antigen
(TAA) positioned amino-terminally relative to the Fc region and/or
carboxy-terminally relative to the CD3 binding region of the
multispecific polypeptide construct.
[0558] A. Design and Generation of Constructs
[0559] Polynucleotides encoding at least a first polypeptide chain
and a second polypeptide chain of the heterodimeric multispecific
polypeptide construct were generated and cloned into a plasmid for
expression. The first polypeptide chain generally included in
order, from the N-terminus to C-terminus, a first Fc polypeptide
(e.g. an Fc hole polypeptide); a non-cleavable linker; and a
variable light (VL) domain of an anti-CD3 antibody. The second
polypeptide chain generally included in order, from the N-terminus
to C-terminus, a second Fc polypeptide (e.g. an Fc knob
polypeptide); the same non-cleavable linker as the first
polypeptide chain; and a variable heavy (VH) domain of an anti-CD3
antibody. The anti-CD3 antibody included either a
disulfide-stabilized (dsFv) antibody (anti-CD3 VH with the mutation
G44C and VL with the mutation G100C) or contained a non-disulfide
stabilized Fv antibody, as set forth in Tables E1.1 and E1.2.
Various exemplary Fc polypeptide pairs to facilitate
heterodimerization of the polypeptide chains were used as set forth
in Tables E1.1 and Table E1.2. One or both of the polypeptide
chains additionally encoded one or more TAA antigen binding domain
amino-terminal to the Fc domain and/or carboxy-terminal to the CD3
binding region, in various configurations. Similar constructs can
be generated using other heterodimeric Fc configurations, including
other knob-into-hole configurations, such as any as described;
other CD3-binding regions, including other anti-CD3 antibodies,
including dsFv or other monovalent fragments; or other TAA
antigen-binding fragments, such as scFv, sdAb or Fab formats can
also be used.
[0560] Among generated constructs, the non-cleavable linker
included linkers ranging from 3-18 amino acids in size. Examples of
non-cleavable linkers used in exemplary generated molecules were
GGS (e.g. contained in exemplary construct cx1356), GGSGGS (SEQ ID
NO:10, contained in exemplary construct cx1357), GGSGGSGGS (SEQ ID
NO:11, contained in exemplary construct cx1358), GGSGGSGGSGGS (SEQ
ID NO:12, contained in exemplary construct cx1359), GGSGGSGGSGGSGGS
(SEQ ID NO:13, contained in exemplary construct cx1360), and
GGGGGSGGGGGSGGGGGS (SEQ ID NO:119, contained in exemplary construct
cx5823 and cx5952) or GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147, contained
in exemplary construct cx681).
[0561] Any antigen binding domain that binds to a TAA can be
employed in the provided multispecific polypeptide constructs.
Exemplary generated proteins contained an antigen binding domain
that binds Folate Receptor Alpha (FR.alpha.), B7H3 (CD276), or
Delta-like 3 (DLL3). The antigen-binding domain can include single
chain fragments (e.g. sdAb or scFv) or two chain antigen-binding
fragments (Fabs). When the TAA was provided as a single chain
fragment, e.g. sdAb or scFv, the TAA antigen binding domain was
linked at the N-terminus to one or both polypeptide chains of the
Fc heterodimer (e.g. hole and/or knob) by a peptide linker, e.g.
PGGGG (SEQ ID NO:102) and/or was linked at the C-terminus to one or
both domains (e.g. VH and/or VL) of the CD3 binding region by a
peptide linker, e.g. GGGG (SEQ ID NO:103). Other similar peptide
linkers can be employed. When the TAA was provided as a Fab
antigen-binding fragment the construct was composed of a VH and CH1
linked directly to one or both Fc polypeptides without a linker, as
well as a light chain composed of a VL and CL. These TAA binding
Fabs can be located on the amino- or carboxy-terminus of the
heterodimeric Fc.
[0562] Multispecific polypeptide constructs were generated
containing 1, 2, 3 or 4 TAA antigen binding domain, such as to
provide for monovalent, bivalent, trivalent, or tetravalent
binding, respectively. In some cases, the TAA antigen binding
domains were the same (mono-epitopic). In some cases, the TAA
antigen binding domains were different, such that the generated
multispecific polypeptide constructs exhibited specificity for at
least two different TAAs, to different epitopes of the same TAA
(bi-epitopic) or the same epitopes of the same TAA
(mono-epitopic).
[0563] Among the generated proteins were constructs in which the
TAA antigen binding domains were composed as single domain
antibodies (sdAbs). Polynucleotides were generated to encode
polypeptide chains of exemplary multispecific polypeptide
constructs containing non-cleavable linkers. These included
constructs designated cx1356, cx1357, cx1358, cx1359, cx1360, and
cx681, targeting FR.alpha. as depicted in FIG. 2B; cx3072, cx5952,
cx6079, cx6080, cx6081, cx5823, cx5873, and cx5965, targeting B7H3
as depicted in FIGS. 3A and 3B; and cx5352, cx5499, cx5800, and
cx5801 targeting DLL3 as depicted in FIGS. 4A and 4B. Notably, some
constructs were generated wherein the VH domain of the dsFv
anti-CD3 antibody and the sdAb were both linked to the same side
(e.g. hole or knob side) of the Fc heterodimer (e.g. cx3072 and
cx5952, shown in FIG. 3A). Constructs were engineered without a
disulfide stabilized Fv or were engineered with a disulfide linkage
stabilizing the VH and VL domains of the anti-CD3 antibody. Some of
the exemplary constructs generated additionally contained a sdAb
(containing a CDR1, a CDR2 and a CDR3 set forth in SEQ ID Nos: 221,
222 and 223, respectively) targeting 4-1BB co-stimulatory receptor
(e.g. cx5823, cx5873, cx5965, cx5352, cx5801, cx5800). A list of
exemplary constrained CD3 binding constructs having sdAb TAA
domains is given below in Table E1.1.
TABLE-US-00008 TABLE E1.1 Exemplary Constrained CD3 engaging
constructs N-term Construct sdAb CD3 Binding C-term sdAb Disulfide
ID Chain (Target) Fc Linker Domain (Target) Stabilized cx681 1
FR.alpha. IgG1-Knob GGSGGGGSGGGGS VH34 (SEQ ID FR.alpha. sdAb2 yes
sdAb1 (SEQ ID GGGGS (SEQ ID NO: 198) (SEQ ID (SEQ ID NO: 82, 86 NO:
147) NO: 121) NO: 120) or 201) 2 FR.alpha. IgG1-Hole GGSGGGGSGGGGS
VL21 (SEQ ID FR.alpha. sdAb2 sdAb1 (SEQ ID GGGGS (SEQ ID NO: 200)
(SEQ ID (SEQ ID NO: 83, 87 NO: 147) NO: 121) NO: 120) or 202)
cx1356 1 FR.alpha. IgG1-Knob GGS VH34 (SEQ ID FR.alpha. sdAb2 yes
sdAb1 (SEQ ID NO: 198) (SEQ ID (SEQ ID NO: 82, 86 NO: 121) NO: 120)
or 201) 2 FR.alpha. IgG1-Hole GGS VL21 (SEQ ID FR.alpha. sdAb2
sdAb1 (SEQ ID NO: 200) (SEQ ID (SEQ ID NO: 83, 87 NO: 121) NO: 120)
or 202) cx1357 1 FR.alpha. IgG1-Knob GGSGGS (SEQ ID VH34 (SEQ ID
FR.alpha. sdAb2 yes sdAb1 (SEQ ID NO: 10) NO: 198) (SEQ ID (SEQ ID
NO: 82, 86 NO: 121) NO: 120) or 201) 2 FR.alpha. IgG1-Hole GGSGGS
(SEQ ID VL21 (SEQ ID FR.alpha. sdAb2 sdAb1 (SEQ ID NO: 10) NO: 200)
(SEQ ID (SEQ ID NO: 83, 87 NO: 121) NO: 120) or 202) cx1358 1
FR.alpha. IgG1-Knob GGSGGSGGS (SEQ VH34 (SEQ ID FR.alpha. sdAb2 yes
sdAb1 (SEQ ID ID NO: 11) NO: 198) (SEQ ID (SEQ ID NO: 82, 86 NO:
121) NO: 120) or 201) 2 FR.alpha. IgG1-Hole GGSGGSGGS (SEQ VL21
(SEQ ID FR.alpha. sdAb2 sdAb1 (SEQ ID ID NO: 11) NO: 200) (SEQ ID
(SEQ ID NO: 83, 87 NO: 121) NO: 120) or 202) cx1359 1 FR.alpha.
IgG1-Knob GGSGGSGGSGGS VH34 (SEQ ID FR.alpha. sdAb2 yes sdAb1 (SEQ
ID (SEQ ID NO: 12) NO: 198) (SEQ ID (SEQ ID NO: 82, 86 NO: 121) NO:
120) or 201) 2 FR.alpha. IgG1-Hole GGSGGSGGSGGS VL21 (SEQ ID
FR.alpha. sdAb2 sdAb1 (SEQ ID (SEQ ID NO: 12) NO: 200) (SEQ ID (SEQ
ID NO: 83, 87 NO: 121) NO: 120) or 202) cx1360 1 FR.alpha.
IgG1-Knob GGSGGSGGSGGSG VH34 (SEQ ID FR.alpha. sdAb2 yes sdAb1 (SEQ
ID GS NO: 198) (SEQ ID (SEQ ID NO: 82, 86 (SEQ ID NO: 13) NO: 121)
NO: 120) or 201) 2 FR.alpha. IgG1-Hole GGSGGSGGSGGSG VL21 (SEQ ID
FR.alpha. sdAb2 sdAb1 (SEQ ID GS NO: 200) (SEQ ID (SEQ ID NO: 83,
87 (SEQ ID NO: 13) NO: 121) NO: 120) or 202) cx5823 1 B7H3 xELL-
GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb5 yes sdAb4 Knob (SEQ GGGGS
(SEQ ID NO: 44) (SEQ ID (SEQ ID ID NO: 88) NO: 119) NO: 216) NO:
214) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID Co-stim (SEQ ID
GGGGS (SEQ ID NO: 72) Receptor NO: 93) NO: 119) sdAb cx5952 1 B7H3
xELL- GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb5 yes sdAb4 Knob (SEQ
GGGGS (SEQ ID NO: 44) (SEQ ID NO: (SEQ ID ID NO: 88) NO: 119) 216)
NO: 214) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID None (SEQ ID
GGGGS (SEQ ID NO: 72) NO: 93) NO: 119) cx6079 1 B7H3 Fc-Het-1
GGGGSGGGGSGGG VH32 (SEQ ID None no sdAb4 (SEQ ID GS NO: 196) (SEQ
ID NO: 194) (SEQ ID NO: 170) NO: 214) 2 B7H3 Fc-Het-2 GGGGSGGGGSGGG
VL20 (SEQ ID None sdAb4 (SEQ ID GS NO: 199) (SEQ ID NO: 195) (SEQ
ID NO: 170) NO: 214) cx6080 1 B7H3 Fc-Het-1 GGGGSGGGGSGGG VH33 (SEQ
ID None yes sdAb4 (SEQ ID GS NO: 197) (SEQ ID NO: 194) (SEQ ID NO:
170) NO: 214) 2 B7H3 Fc-Het-2 GGGGSGGGGSGGG VL21 (SEQ ID None sdAb4
(SEQ ID GS NO: 200) (SEQ ID NO: 195) (SEQ ID NO: 170) NO: 214)
cx6081 1 B7H3 Fc-Het-1 GGGGSGGGGSGGG VH13 (SEQ ID None yes sdAb4
(SEQ ID GS NO: 44) (SEQ ID NO: 194) (SEQ ID NO: 170) NO: 214) 2
B7H3 Fc-Het-2 GGGGSGGGGSGGG VL10 (SEQ ID None sdAb4 (SEQ ID GS NO:
72) (SEQ ID NO: 195) (SEQ ID NO: 170) NO: 214) cx5841 1 B7H3 xELL-
GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb3 yes sdAb4 Knob (SEQ GGGGS
(SEQ ID NO: 44) (SEQ ID (SEQ ID ID NO: 84 NO: 119) NO: 217) NO:
214) 88 or 203) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID Co-stim
(SEQ ID GGGGS (SEQ ID NO: 72) Receptor NO: 91, 93 NO: 119) sdAb or
206) cx5187 1 B7H3 xELL- GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb3 yes
sdAb4 Knob (SEQ GGGGS (SEQ ID NO: 44) (SEQ ID (SEQ ID ID NO: 84,
NO: 119) NO: 217) NO: 214) 88 or 203) 2 None xELL-Hole
GGGGGSGGGGGSG VL10 (SEQ ID Co-stim (SEQ ID GGGGS (SEQ ID NO: 72)
Receptor NO: 91, 93 NO: 119) sdAb or 206) cx5499 1 DLL3 xELL-
GGGGGSGGGGGSG VH13 (SEQ ID DLL3 sdAb2 yes sdAb1 Knob (SEQ GGGGS
(SEQ ID NO: 44) (SEQ ID NO: (SEQ ID ID NO: 84, NO: 119) 220) NO:
219) 88 or 203) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID None
(SEQ ID GGGGS (SEQ ID NO: 72) NO: 91, 93 NO: 119) or 206) cx3072 1
B7H3 IgG1-Knob GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb1 yes sdAb2 (SEQ
ID GGGGS (SEQ ID NO: 44) (SEQ ID (SEQ ID NO: 82, 86 NO: 119) NO:
218) NO: 215) or 201) 2 None IgG1-Knob GGGGGSGGGGGSG VL10 (SEQ ID
None (SEQ ID GGGGS (SEQ ID NO: 72) NO: 83, 87 NO: 119) or 202)
cx5873 1 None xELL- GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb3 yes Knob
(SEQ GGGGS (SEQ ID NO: 44) (SEQ ID ID NO: 84, NO: 119) NO: 217) 88
or 203) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID Co-stim (SEQ ID
GGGGS (SEQ ID NO: 72) Receptor NO: 91, 93 NO: 119) sdAb or 206)
cx5965 1 B7H3 xELL- GGGGGSGGGGGSG VH13 (SEQ ID none yes sdAb4 Knob
(SEQ GGGGS (SEQ ID NO: 44) (SEQ ID ID NO: 84, NO: 119) NO: 214) 88
or 203) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID Co-stim (SEQ ID
GGGGS (SEQ ID NO: 72) Receptor NO: 91, 93 NO: 119) sdAb or 206)
cx5352 1 DLL3 xELL- GGGGGSGGGGGSG VH13 (SEQ ID DLL3 sdAb2 yes sdAb1
Knob (SEQ GGGGS (SEQ ID NO: 44) (SEQ ID NO: (SEQ ID ID NO: 84, NO:
119) 220) NO: 219) 88 or 203) 2 None xELL-Hole GGGGGSGGGGGSG VL10
(SEQ ID Co-stim (SEQ ID GGGGS (SEQ ID NO: 72) Receptor NO: 91, 93
NO: 119) sdAb or 206) cx5800 1 DLL3 xELL- GGGGGSGGGGGSG VH13 (SEQ
ID None yes sdAb1 Knob (SEQ GGGGS (SEQ ID NO: 44) (SEQ ID ID NO:
84, NO: 119) NO: 219) 88 or 203) 2 None xELL-Hole GGGGGSGGGGGSG
VL10 (SEQ ID Co-stim (SEQ ID GGGGS (SEQ ID NO: 72) Receptor NO: 91,
93 NO: 119) sdAb or 206) cx5801 1 None xELL- GGGGGSGGGGGSG VH13
(SEQ ID DLL3 sdAb2 yes Knob (SEQ GGGGS (SEQ ID NO: 44) (SEQ ID NO:
ID NO: 84, NO: 119) 220) 88 or 203) 2 None xELL-Hole GGGGGSGGGGGSG
VL10 (SEQ ID Co-stint (SEQ ID GGGGS (SEQ ID NO: 72) Receptor NO:
91, 93 NO: 119) sdAb or 206)
[0564] Exemplary generated constructs also included constructs in
which the TAA antigen binding domains were composed as a Fab
(designated MAB formats). In this example, an anti-B7H3 Fab was
used composed of a heavy chain Fd set forth in SEQ ID NO:127 and a
light chain set forth in SEQ ID NO:128. Polynucleotides were
generated to encode polypeptide chains of exemplary multispecific
polypeptide constructs containing non-cleavable linkers. These
included cx5067, cx6083, and cx6084, as depicted in FIG. 3C.
Constructs were engineered without a disulfide stabilized anti-CD3
antibody Fv or were engineered with a disulfide linkage stabilizing
the VH and VL domains of the anti-CD3 antibody (anti-CD3 VH with
the mutation G44C and VL with the mutation G100C). A list of
exemplary Fab constructs is provided below in Table E1.2.
TABLE-US-00009 TABLE E1.2 Exemplary B7H3-targeted constrained CD3
engaging constructs Construct N-term CD3 Binding C-term sdAb
Disulfide ID Chain Domain Fc Linker Domain (Target) Stabilized
cx5067 1 B7H3-Fab Fc-Het-1 GGGGSGGGGSGGGG VH32 (SEQ ID None no (SEQ
ID (SEQ ID S NO: 196) NOs: 127, NO: 194) (SEQ ID NO: 170) 128) 2
B7H3-Fab Fc-Het-2 GGGGSGGGGSGGGG VL20 (SEQ ID None (SEQ ID (SEQ ID
S NO: 199) NOs: 127, NO: 195) (SEQ ID NO: 170) 128) cx6083 1
B7H3-Fab Fc-Het-1 GGGGSGGGGSGGGG VH33 (SEQ ID None yes (SEQ ID (SEQ
ID S NO: 197) NOs: 127, NO: 194) (SEQ ID NO: 170) 128) 2 B7H3-Fab
Fc-Het-2 GGGGSGGGGSGGGG VL21 (SEQ ID None (SEQ ID (SEQ ID S NO:
200) NOs: 127, NO: 195) (SEQ ID NO: 170) 128) cx6084 1 B7H3-Fab
Fc-Het-1 GGGGSGGGGSGGGG VH13 (SEQ ID None yes (SEQ ID (SEQ ID S NO:
44) NOs: 127, NO: 194) (SEQ ID NO: 170) 128) 2 B7H3-Fab Fc-Het-2
GGGGSGGGGSGGGG VL10 (SEQ ID None (SEQ ID (SEQ ID S (SEQ ID NO: 170)
NO: 72) NOs: 127, NO: 195) 128)
[0565] B. Expression and Purification of Generated Constructs
[0566] Separate plasmids encoding each chain of the heterodimeric
constrained CD3 binding protein were transiently transfected at an
equimolar ratio into mammalian cells (either HEK293 or CHO) using
polyethylenimine. Recombinant protein secreted into the supernatant
was collected after 3-14 days, and purified by protein A
chromatography, followed by either preparative size exclusion
chromatography (SEC) or flow-through hydrophobic interaction
chromatography (HIC). In some cases, heterodimeric protein was
enriched for during purification due to a mutation designed into
one chain of the heterodimeric Fc at position I253R or H435R (e.g.
in the hole-Fc) such that it did not bind protein A, and thus
homodimers of I253R or H435R were not purified. The second
chromatography step by SEC (AKTA with Superdex-200 resin) or FT-HIC
(AKTA with butyl/phenyl sepharose) was used to remove undesired
cross-paired species containing two heterodimeric Fcs that were
more hydrophobic and twice the expected molecular weight.
[0567] The method favored production of heterodimeric multispecific
polypeptide constructs, containing properly paired species of
heterodimeric Fc and the anti-CD3 Fv (e.g. disulfide stabilized
anti-CD3 Fv). Purified heterodimeric constrained CD3 binding
protein was stable and did not accumulate cross-paired species upon
prolonged incubation at 4.degree. C. or increased protein
concentration.
Example 2: Assessment of Binding of Constrained CD3 Binding
Constructs to Cancer Cells and Primary T Cells by Flow
Cytometry
[0568] This Example describes studies assessing binding of
exemplary constructs to T cells or to cancer cells. These studies
were carried out in single cultures containing either only the T
cells or only the cancer cells in isolation from each other.
[0569] A. FR-Alpha Binding
[0570] Binding of exemplary multispecific polypeptide constructs of
the disclosure containing an antigen-binding domain directed
against Folate Receptor Alpha (FR.alpha.) to CD3 on the surface of
primary T cells and to FR.alpha. expressing cells (Ovcar-5) was
assessed by flow cytometry. The tumor antigen binding domains of
the tested constructs bind the Folate Receptor Alpha (FR.alpha.),
which is not expressed on the primary T cells. The tested
constructs included cx1356 and cx681, containing a non-cleavable
linker of 3 amino acids in cx1356 or a non-cleavable linker of 18
amino acids in cx681 (see FIG. 2B and Table E1.1).
[0571] For the studies described in FIGS. 5A and 5B, 100 nM of each
construct, cx1356 or cx681 was used and assessed for binding to
either the Ovcar-5 cells or primary T-cells. An anti-CD3 antibody
was included as a control. A titration of the constrained CD3
engaging constructs on either FR.alpha. expressing cells (Ovcar-5;
FIG. 5C) or isolated primary T-cells (FR.alpha. negative; FIG. 5D),
was conducted to assess binding. Bound constructs were detected
using fluorophore-conjugated anti-human IgG Fc secondary
antibody.
[0572] The tested FR.alpha.-targeting constructs with various
linkers between the Fc and the component of the CD3 binding domains
were found to bind FR.alpha. expressing cells (Ovcar-5) (FIGS. 5A
and 5C), but lacked the capacity to bind T-cells (FIGS. 5B and 5D).
These results are consistent with a finding that binding to CD3 on
T cells in isolation is constrained in the provided formats.
[0573] B. B7H3 Binding
[0574] Binding of exemplary multispecific constructs containing an
antigen-binding domain directed against B7H3 were assessed for
binding to B7H3 positive A375 tumor cells or primary T-cells. The
constructs were generated containing antigen-binding domain(s) that
were either sdAbs or a FABs, and that were positioned either only
N-terminal to the Fc or both N-terminal to the Fc and C-terminal to
the anti-CD3 binding domain (see FIGS. 3A and 3C and Table E1.1).
Among the various formats of constructs tested included:
sdAb-Fc-dsFV-sdAb (cx3072, cx5952) sdAb-Fc-FV (cx6079),
sdAb-Fc-dsFV (c56080, cx6081), MAB-FV (cx5067) and MAB-dsFV
(cx6083, cx6084), where the FV represents the anti-CD3 binding
domain composed of VH and VL domain pairs and "ds" notes disulfide
stabilized via an engineered interdomain disulfide bond.
[0575] FIGS. 6A-F demonstrate that these constructs are capable of
binding to B7H3 but not T-cells in isolation. Binding was assessed
as described above via flow cytometry using a
fluorophore-conjugated anti-human Fc secondary antibody. cx3072
bound to A375 cells with high affinity (FIG. 6A) but not to
isolated T-cells (FIG. 6B). The tested sdAb-Fc-dsFV-sdAb (cx5952)
displayed higher binding affinity compared to a FAB containing
MAB-FV (cx5067) and MAB-dsFV constructs (cx6083, cx6084) (FIG. 6C).
The B7H3-targeting sdAb containing constructs (cx5952, cx6079,
cx6080, and cx6081) bound to B7H3 positive cells with similar
affinities (depicted in FIG. 6E), with cx5952 displaying higher
maximal binding. cx6079, cx6080 and cx6081 contain two identical
B7H3-targeting sdAbs, whereas cx5952, and cx3072 contain two
distinct B7H3-targeting sdAb that bind different epitopes. The
MAB-FV, cx5067, contains two identical B7H3-targeting FAB domains.
Notably none of the exemplary B7H3-targeted constrained CD3
engaging constructs bound isolated primary human T-cells, as
depicted in FIGS. 6B, 6D, and 6F. These results further support
that binding to CD3 on T cells in isolation is constrained in the
provided formats.
Example 3: Assessment of CD3 Signaling Activity in Co-Cultures with
Antigen-Expressing Target Cells and Impact of Linker Length on
Activity
[0576] The effect of various length linkers between the Fc and the
component domains (VH and VL) that comprise the CD3 binding region
on T-cell activating capacity was tested using a Jurkat reporter
assay. The CD3 reporter cells were developed from Jurkat cells that
naturally express CD3 and were engineered to express NFAT-driven
green fluorescence protein (GFP). Agonism of CD3 results in NFAT
signaling and production of green fluorescence.
[0577] Antigen targeting constrained CD3 engaging constructs were
titrated onto co-cultures of target cells and Jurkat CD3 reporter
cells. In this assay, target cells included either IGROV1
(FR.alpha. positive) or NCI-460 (FR.alpha. negative). For reporter
assays utilizing adherent antigen expressing target cells, target
cells were seeded, allowed to settle at room temperature for
uniform distribution, and incubated for several hours at 37.degree.
C. to permit adherence prior to addition of reporter cells and
antigen targeting constrained CD3 engaging constructs. Assay plates
were serially imaged using an IncuCyte ZOOM system and CD3 reporter
cell activation was determined by measuring GFP expression as the
total integrated green object in the well.
[0578] FR.alpha.-targeting constrained CD3 engaging constructs,
generated as described in Example 1 containing GlySer-based linkers
of varying lengths as listed in Table E3 were used in these
assays.
TABLE-US-00010 TABLE E3 Tested Linker Lengths SEQ ID NO Linker --
gs3: GGS 10 gs6: GGSGGS 11 gs9: GGSGGSGGS 12 gs12: GGSGGSGGSGGS 13
gs15: GGSGGSGGSGGSGGS 119 gs18: GGGGGSGGGGGSGGGGGS 147 gs18:
GGSGGGGSGGGGSGGGGS
[0579] As shown in FIGS. 7A-7F, the T-cell activating activity as
determined by fluorescent intensity was dependent on co-culture
with FR.alpha.-antigen expressing target cells. The length of the
linker and T-cell activating capacity were positively correlated.
T-cell activating capacity was shown to directly relate to linker
length, indicating shorter linkers restrict CD3 binding to a
greater extent (see FIGS. 7A, 7C and 7E). Importantly, T-cell
engagement of the constructs is dependent on TAA-binding, as these
constructs did not demonstrate a T-cell binding capacity in
isolation (e.g. solution form when unbound to target TAA) as shown
above in Example 2. Further, no observable fluorescence was
measured in co-cultures with FR.alpha. negative cells (FIGS. 7B, 7D
and 7E). Together, these constructs displayed restricted or
substantially reduced binding to CD3, yet were capable of
activating T-cells in a target dependent manner.
Example 4: Assessment of T Cell Activating Activity of
B7I13-Targeted Constrained CD3 Binding Constructs
[0580] Constructs containing either a B7H3-targeted sdAb or a Fab
as the tumor-associated antigen-binding domain were assessed for
T-cell activating activity in a T cell reporter assay and in a T
cell cytotoxicity assay. Activity of B7H3-targeted constrained CD3
engaging constructs that were formatted with an anti-B7H3 sdAb
(e.g. cx5823, cx6079, cx6080, cs6081, cx3072 and cx5952) or
anti-B7H3 MAB constructs formatted with a Fab (e.g. cx5067, cx6083
or cx6084) as the antigen-binding domain(s) were assessed (see
FIGS. 3A-3C and Table E1.1). All tested constructs, except cx5067
and cx6079, contained a disulfide-stabilized anti-CD3 Fv (dsFv)
containing an interchain disulfide bond created by the modification
of anti-CD3 VH G44C paired with VL G100C. The anti-CD3 Fv of
cx5067, designated MAB-Fv, was not disulfide-stabilized.
[0581] A. T cell Reporter Activity
[0582] The NFAT-GFP CD3 Jurkat reporter described in Example 3 was
used to compare the CD3 agonistic properties of B7H3-targeted
constrained CD3 engaging constructs when co-cultured in the
presence of B7H3-positive cells (A375) or non-target CCRF-CEM cells
that naturally lack B7H3 expression. In this assay, anti-B7H3 sdAb
constructs, cx5823, cx6079, cx6080 and cx6081, or anti-B7H3 Fabs
constructs, cx5067, cx6083 and cx6084, were used as the
B7H3-targeting domains. As shown in FIG. 8A, the constructs
containing B7H3-targeted sdAb displayed similar potencies of
antigen-dependent CD3 activation. As shown in FIG. 8C the exemplary
cx5823 construct containing B7H3-targeted sdAbs was found to be
superior at mediating antigen-dependent CD3 activation compared to
the constructs containing B7H3-targeted Fabs. Although cx5823 is
formatted with a binding domain for a costimulatory receptor, it is
unlikely that this component contributed to the difference in
results, since Jurkat T cells do not express the costimulatory
receptor. None of the constructs demonstrated activity against the
B7H3-negative CCRF-CEM cells (FIGS. 8B and 8D).
[0583] B. Cytotoxicity
[0584] To further assess activity of the molecules, exemplary
B7H3-targeted constructs cx3072 and cx5952 (each formatted as
sdAb-dsFv), cx6083 and cx6084 (MAB-dsFv), cx5067 (MAB-Fv), cx6079
(sdAb-Fv), and cx6080 and cx 6081 (sdAb-dsFv) were tested in a
T-cell-mediated cytotoxicity assay. Target cells included the B7H3
positive cell line, A375, and either modified A375 cells, wherein
B7H3 gene was disrupted by CRISPR (A375:B7H3 KD), or CCRF-CEM cells
that naturally lacked B7H3 expression. Target cells were seeded at
1.0.times.10.sup.4 cells per well, allowed to settle at room
temperature for uniform distribution, and incubated for several
hours at 37.degree. C. Primary T cells were negatively enriched
from PBMCs isolated from healthy human donor leukopaks and added at
a 10:1 T cell-to-target cell ratio. Green caspase-3/7 reagent was
added, which fluorescently labels nuclear DNA of cells undergoing
apoptosis was added. Multispecific constructs with constrained CD3
engaging activity were titrated onto the co-culture and assay
plates were serially imaged using an IncuCyte ZOOM system. Target
cell death was determined by measuring total red/green overlap
object area.
[0585] As shown in FIGS. 9A and 9B, exemplary constructs cx3072 and
cx5952 containing sdAb B7H3-targeted antigen-binding domains
induced potent T-cell-mediated cytotoxicity of B7H3 positive (A375)
but not B7H3 negative cell lines.
[0586] When compared to exemplary B7H3-targeting constrained CD3
engagers with Fab B7H3-targeting domains (cx5067, cx6083 and
cx6084), the exemplary cx5952 sdAb B7H3-targeting constrained CD3
engager mediated enhanced target-dependent T-cell cytotoxicity
(FIG. 10A). No measurable T cell cytotoxicity was observed against
the B7H3 negative cell line CCRF-CEM for any of the tested
constructs, consistent with the capacity to potently induce
antigen-dependent T-cell activation (FIG. 10B). Of the constructs
tested, representative MAB-dsFV constructs cx6084 and cx6083
contained the engineered disulfide, whereas the representative
MAB-FV construct cx5067 lacked this stabilizing modification.
Notably cx6083 and cx5067 are identical with the exception of the
presence (cx6083) or absence (cx5067) of the engineered disulfide
within the anti-CD3 FV domain (depicted in FIG. 3C). The engineered
disulfide was created by the modification of G44C within VH and
G100C within VL. As shown in FIG. 10A, cx6083 displayed superior
potency in mediating target-dependent T-cell cytotoxicity compared
to cx5067, suggesting that the incorporation of the inter-domain
disulfide is beneficial in T-cell mediated cytotoxicity, likely by
enhancing proper association of the VH and VL domains that comprise
the anti-CD3 FV.
[0587] When compared to other exemplary B7H3-targeting constrained
CD3 engagers with sdAb B7H3-targeting domains (cx6079, cx6080 and
cx6081), the exemplary cx5952 sdAb B7H3-targeting constrained CD3
engager mediated enhanced target-dependent T-cell cytotoxicity
(FIG. 10C). No measurable T cell cytotoxicity was observed against
the B7H3 negative cell line CCRF-CEM for any of the tested
constructs, consistent with the capacity to potently induce
antigen-dependent T-cell activation (FIG. 10D). Of the constructs
tested, sdAb-dsFV constructs cx5952, cx6080 and cx6081 contained
the engineered disulfide linkage, whereas the sdAb-FV construct
cx6079 lacked this stabilizing modification. The engineered
disulfide was created by the modification of G44C within VH and
G100C within VL. Notably, cx5952 was engineered to have two
distinct B7H3 targeting domains, one located at the amino terminal
and one located at the carboxy terminal. cx6079, cx6080, and cx6081
were engineered to have two identical B7H3 targeting domains, both
located at the amino terminal (see FIG. 3A).
[0588] C. T Cell Modulation
[0589] To further assess T cell modulation, exemplary multispecific
CD3 constrained binding constructs was assessed by monitoring the
ability of the constructs to modulate T cell activation markers. To
assess T cell activation, suspension cells from T cell cytotoxicity
assays above, involving culture of T cells with B7H3 positive
(A375) or B7H3 negative cell lines (CCRF-CEM) in the presence of an
exemplary B7H3-targeted constrained CD3 engaging construct, cx5952,
were collected. Cells were stained with a live/dead stain and
fluorophore-conjugated anti-CD4, anti-CD8, anti-CD25, anti-CD69,
and/or anti-CD71 antibodies. Cells were analyzed using a SONY
SA3800 spectral analyzer and CD4+ or CD8+ T cell activation was
determined by measuring expression levels of CD25, CD69 or CD71 or
percent CD25-, CD69- or CD71-positive.
[0590] Results are shown for CD25 expression (FIG. 11A), CD69
expression (FIG. 11B) and CD71 expression (FIG. 11C) on CD4+ and
CD8+ T cells following the co-culture with B7H3 positive (A375) or
B7H3 negative cell lines (CCRF-CEM) in the presence of cx5952. The
results showed that cx5952 mediated a dose-dependent B7H3-dependent
T-cell activation via CD3 binding, as evidenced by increased
expression of CD25, CD69 and CD71 in CD4+ and CD8+ T cells.
[0591] When compared to the other exemplary B7H3-targeting
constrained CD3 engagers with sdAb B7H3-targeting domains (cx6079,
cx6080 and cx6081), the exemplary cx5952 sdAb B7H3-targeting
constrained CD3 engager mediated increased T cell activation as
evidenced by increased expression of CD25 in CD4+ T cells (FIG.
11D) and in CD8+ T cells (FIG. 1111) and increased expression of
CD71 in CD4+ T cells (FIG. 11F) and in CD8+ T cells (FIG. 11J).
Increased expression of the surface markers on T cells was not
observed in the presence of the B7H3-targeting constrained CD3
engager constructs in cultures with B7H3 negative cell lines (FIGS.
11E and 11G for CD4+ T cells and FIGS. 11I and 11K for CD8+ T
cells).
[0592] D. T Cell Cytokine Production
[0593] Supernatants from T cell cytotoxicity tumor cell co-culture
assays, involving co-culture of T cells with B7H3 positive, A375 or
negative, CCRF-CEM cells in the presence of cx5952, cx6083, cx6084
or cx5067, were analyzed for IFN.gamma. content by sandwich ELISA.
A standard curve was generated from which cytokine concentration
values of supernatant samples were interpolated. Samples that had
absorbance values below the lower limit of detection were assigned
a cytokine concentration equal to half that of the lowest standard
concentration. As shown in FIG. 12A, the representative
sdAb-Fc-dsFV-sdAb construct, cx5952, was superior to the tested
B7H3-targeted FAB containing constructs, cx6083, cx6084 and cx5067
at eliciting target-dependent cytokine release from activated
T-cells. Importantly, the MAB-dsFV constructs, cx6083 and cx6084
were superior to the MAB-FV construct, cx5067, demonstrating the
importance of interdomain disulfide stabilizing modification for
enhancing T-cell function.
[0594] When compared to the other exemplary B7H3-targeting
constrained CD3 engagers with sdAb B7H3-targeting domains (cx6079,
cx6080 and cx6081), the exemplary cx5952 sdAb B7H3-targeting
constrained CD3 engager mediated substantially increased production
of IFN.gamma. in the presence of B7H3-target cells T cells but not
in cultures with B7H3 negative cell lines (FIG. 12B)
[0595] E. Summary
[0596] These observations further support that the antigen-targeted
constrained CD3 format provided herein lack or exhibit reduced
T-cell binding in isolation while maintaining potent B7H3-dependent
T-cell cytotoxicity inducing capacities. Without wishing to be
bound by theory, together these results show that utilization of
antigen targeted sdAbs instead of a Fabs may reduce the immune
synapse distance between the TAA expressing tumor cell and the CD3
expressing T-cells and enhance T cell activity and cytotoxicity.
Notably, it was found that the inclusion of an interchain disulfide
bond created by the modification of anti-CD3 VH G44C paired with VL
G100C greatly enhanced the activity of constrained CD3 engaging
constructs. Further, the more potent B7H3-dependent T cell activity
by cx5952 compared to other sdAb B7H3-targeting domain constructs
suggests that the positioning of the B7H3-targeting sdAb C-terminal
to the anti-CD3 binding domain or the fact that cx5952 binds two
distinct epitopes on B7H3 whereas the other constructs tested bind
to a single epitope in a bivalent manner, contributed to this
enhanced activity.
Example 5: Assessment of CD3-Constrained Multispecific Constructs
Containing Single or Multiple B7H3-Binding Targeting Domains
[0597] Activity of constructs containing a monovalent sdAb
antigen-binding domain (positioned at either the N or C-terminus)
was compared to activity of dual binding (bivalent) constructs that
contained antigen-targeting sdAbs positioned at both the N and
C-termini. Binding was assessed substantially as described in
Example 2 and T cell activity was assessed in the Jurkat reporter
assay and T cell cytotoxicity assays substantially as described in
Examples 3 and 4.
[0598] A. Binding
[0599] As shown in FIG. 13A the bivalent B7H3-targeting constrained
CD3 engaging constructs, cx5187 and cx5823, displayed higher
affinity binding to B7H3 positive A375 cells, compared to the
monovalent versions, cx5873 and cx5965. None of these constructs
displayed any detectable binding to B7H3 negative CCRF-CEM cells or
isolated T-cells (FIG. 13B).
[0600] B. T Cell Reporter Activity
[0601] B7H3 Antigen-dependent CD3 agonistic capacities of
antigen-targeted constrained CD3 engaging constructs that engage
the antigen in a monovalent or bivalent manner were assessed using
CD3-NFAT Jurkat reporter cells, in an assay substantially as
described above. As shown in FIG. 13C, substantially increased
fluorescence reporter activity was observed in the presence of the
exemplary bivalent B7H3-targeted construct cx5187 compared to
reporter activity for the exemplary monovalent constructs cs5873
and cx5965. No reporter activity was observed when constructs were
incubated with Jurkat reporter cells co-cultured with B7H3-negative
CCRF target cells (FIG. 13D).
[0602] C. Cytotoxic Activity
[0603] Cytotoxicity of B7H3-targeted CD3 constrained binding
constructs was assessed against a melanoma cell line, A375, and a
T-cell acute lymphoblastic leukemia cell line, CCRF-CEM, which were
used as B7H3 positive and negative cell lines, respectively.
Cytotoxicity was assessed substantially as described in Example 4.
As shown in FIG. 14A an exemplary bivalent B7H3-targeted
constrained CD3 engaging construct, cx5187, displayed enhanced
target-dependent T-cell mediated cytotoxicity compared to the
monovalent versions of the constructs, cx5873 and cx5965. In these
assays, no cytotoxicity was observed in the absence of B7H3
expression of the target cells, as shown in FIG. 14B wherein the
CCRF-CEM cells were used as target cells.
[0604] D. T Cell Modulation
[0605] T cell modulation was assessed by monitoring expression of
CD25, substantially as described in Example 4, in suspension cells
from T cell cytotoxicity assays above, involving culture of T cells
with B7H3 positive (A375) or B7H3 negative cell lines (CCRF-CEM) in
the presence of cx5187, cx5873 or cx5965. As shown in FIGS. 15A and
15B, an exemplary bivalent B7H3-targeted constrained CD3 engaging
construct, cx5187, displayed enhanced target-dependent T-cell
mediated activation compared to the monovalent versions of the
constructs, cx5873 and cx5965, as evidenced by enhanced potency of
CD25 upregulation on CD4 and CD8 T-cells. In these assays, no
T-cell activation was observed in the absence of B7H3 expression of
the target cells, as shown in FIGS. 15C and 15D, wherein the
CCRF-CEM cells were used as target cells. These results
demonstrated that the B7H3-targeting constrained CD3 engaging
constructs induced potent antigen-dependent activation of both CD4
and CD8 T-cells.
[0606] D. Summary
[0607] Together, these results demonstrate that bivalent
antigen-targeted constrained CD3 engaging constructs displayed
superior antigen-dependent CD3 binding and activity than the
monovalent antigen-targeted constrained CD3 engaging constructs.
These results are consistent with a finding that constructs
containing dual antigen-binding domains positioned at both the N
and C-termini have superior binding and T cell activity than
monovalent constructs containing only a single monovalent
antigen-binding domain. Furthermore, without wishing to be bound by
theory, positioning one of the sdAbs C-terminal to the CD3 binding
domain may form a more optimal immune synapse compared to
constructs wherein the sdAbs are only positioned N-terminal to the
Fc as the latter may increase the immune synapse distance.
Example 6: Assessment of a CD3-Constrained Multispecific Constructs
Containing B7I13-Targeting sdAb and Fab Domains
[0608] Constructs containing either B7H3-targeted sdAb(s) or a Fab
as the tumor-associated antigen-binding domain were assessed for
T-cell activating activity. Activity of B7H3-targeted constrained
CD3 engaging constructs that were formatted with anti-B7H3 sdAbs
(e.g. cx5952 and cx6079) or anti-B7H3 MAB constructs formatted with
a Fab (e.g. cx5067, cx6083 or cx6084) as the antigen-binding
domain(s) were assessed (see FIGS. 3A and 3C and Table E1.1 and
E1.2). All tested constructs, except cx6079 and cx5067, contained a
disulfide-stabilized anti-CD3 Fv (dsFv) containing an interchain
disulfide bond created by the modification of anti-CD3 VH G44C
paired with VL G100C. The anti-CD3 Fv of cx5067, designated MAB-Fv,
and the anti-CD3 Fv of cx6079, designated sdAb-Fc-Fv, were not
disulfide-stabilized. Additionally, cx5952 was engineered to
contain two distinct B7H3-targeting sdAb domains, with one located
N-terminal to the Fc domain and one located C-terminal to the
CD3-binding domain. By contrast, cx6079 was engineered to contain
two identical B7H3-targeting sdAb domains, both located N-terminal
to the Fc domain. The Fvs of all three Fab constructs were
engineered to be N-terminal to the Fc domain.
[0609] A. Cytotoxicity
[0610] Cytotoxicity of B7H3-targeted CD3 constrained binding
constructs was assessed substantially as described in Example 4.
Cytotoxicity was assessed against a melanoma cell line, A375, and a
T-cell acute lymphoblastic leukemia cell line, CCRF-CEM, which were
used as B7H3 positive and negative cell lines, respectively. As
shown in FIG. 16A the exemplary constrained CD3 engaging constructs
formatted with B7H3-targeting sdAbs, cx5952 and cx6079, were
superior at eliciting antigen-dependent T-cell cytotoxicity
compared to the anti-B7H3 MAB constructs formatted with a Fab,
cx5067, cx6083, and cx6084. Notably, cx5952 was more potent than
cx6079, suggesting the positioning of the B7H3-targeting sdAb
C-terminal to the anti-CD3 binding domain and/or the stabilization
of the anti-CD3 FV via engineered disulfide contributed to this
enhanced activity. In these assays, no cytotoxicity was observed in
the presence of B7H3-negative CRF-CEM cells target cells, as shown
in FIG. 16B.
[0611] B. T Cell Modulation
[0612] To further assess T cell modulation, exemplary multispecific
CD3 constrained binding constructs were assessed by monitoring the
ability of the constructs to modulate T cell activation markers,
substantially as described in Example 4. To assess T cell
activation, suspension cells from T cell cytotoxicity assays above,
involving culture of T cells with B7H3 positive (A375) or B7H3
negative cell lines (CCRF-CEM) in the presence of an exemplary
B7H3-targeted constrained CD3 engaging constructs, were collected.
Tested constructs included anti-B7H3 constructs formatted with
sdAbs (e.g. cx5952 and cx6079) and anti-B7H3 constructs formatted
with a Fab (e.g. cx5067, cx6083 and cx6084)
[0613] Cells were stained with a live/dead stain and
fluorophore-conjugated anti-CD4, anti-CD8, anti-CD25 and/or
anti-CD71 antibodies. Cells were analyzed using a SONY SA3800
spectral analyzer and CD4+ or CD8+ T cell activation was determined
by measuring expression levels of CD25 or CD71 or percent CD25- or
CD71-positive.
[0614] Results are shown for CD25 expression (FIG. 16C-F) and CD71
expression (FIG. 16G-J) on CD4+ and CD8+ T cells following the
co-culture with B7H3 positive (A375) or B7H3 negative cell lines
(CCRF-CEM) in the presence of the described constructs. The results
showed that cx5952 mediated a dose-dependent B7H3-dependent T-cell
activation via CD3 binding, as evidenced by increased expression of
CD25 and CD71 in CD4+ and CD8+ T cells. cx5952 was the most potent
over other B7H3-targeted constrained CD3 engaging constructs and
inducing T-dependent T-cell activation.
[0615] C. T Cell Cytokine Production
[0616] Supernatants from T cell cytotoxicity tumor cell co-culture
assays, involving co-culture of T cells with B7H3 positive, A375 or
negative, CCRF-CEM cells in the presence of cx5952, cx6079, cx6083,
cx6084 or cx5067, were analyzed for IFN.gamma. content by sandwich
ELISA. A standard curve was generated from which cytokine
concentration values of supernatant samples were interpolated.
Samples that had absorbance values below the lower limit of
detection were assigned a cytokine concentration equal to half that
of the lowest standard concentration. As shown in FIG. 16K, the
representative sdAb-Fc-dsFV-sdAb construct, cx5952, was superior to
the tested B7H3-targeted FAB containing constructs, cx6083, cx6084
and cx5067 at eliciting target-dependent cytokine release from
activated T-cells. This is consistent with the finding from the
antigen dependent cytotoxicity and activation assays. Importantly,
the MAB-dsFV constructs, cx6083 and cx6084 were superior to the
MAB-FV construct, cx5067, demonstrating the importance of
interdomain disulfide stabilizing modification for enhancing T-cell
function.
[0617] D. Summary
[0618] Together, these results demonstrate that constrained
anti-CD3 constructs formatted with anti-B7H3 sdAb binding domains
were superior at eliciting antigen-dependent T-cell cytotoxicity
compared to the anti-B7H3 MAB constructs formatted with a Fab B7H3
binding domain. Further, that cx5952 was more potent than cx6079
suggested that the positioning of the B7H3-targeting sdAb
C-terminal to the CD3 binding domain, the stabilization of the
anti-CD3 FV via engineered disulfide, or both, contribute to
enhanced activity. Without wishing to be bound by theory,
positioning one of the sdAbs C-terminal to the CD3 binding domain
may form a more optimal immune synapse compared to constructs
wherein the sdAbs are only positioned N-terminal to the Fc as the
latter may increase the immune synapse distance.
Example 7: Assessment of CD3-Constrained Multispecific Constructs
Containing Single or Multiple Antigen-Binding DLL3-Targeting
Domains
[0619] This example describes the assessment and characterization
of exemplary generated DLL3-targeted constrained CD3 engaging
constructs in human primary T cell in vitro assays.
[0620] Binding and activity of DLL3-targeted constrained CD3
engaging constructs that were formatted with an anti-DLL3 sdAb
(e.g. cx5352, cx5800, cx5801, and cx5499) as the antigen-binding
domain(s) were assessed (see FIGS. 4A-4B and Table E1.1). All
tested constructs contained a disulfide-stabilized anti-CD3 Fv
(dsFv) containing an interchain disulfide bond created by the
modification of anti-CD3 VH G44C paired with VL G100C. Further, the
DLL-3-targeted constructs were engineered to contain a
co-stimulatory receptor sdAb C-terminal to the CD3 dsFv, except for
cx5499, which did not contain this co-stimulatory receptor sdAb
domain.
[0621] A. Binding
[0622] Binding was assessed substantially as described in Example
2. As shown in FIG. 17A the bivalent DLL3-targeting constrained CD3
engaging constructs, cx5352 displayed higher affinity binding to
DLL3 positive, SHP-77 cells compared to the monovalent versions,
cx5800 and cx5801. None of the constructs tested displayed binding
to DLL3-negative primary T cells, as depicted in FIG. 17B. These
binding assays were conducted by flow cytometry, wherein bound
constructs were detected using a fluorophore-conjugated anti-human
IgG Fc secondary antibody.
[0623] A. T Cell Reporter Activity
[0624] T cell activity was assessed in a reporter assay
substantially as described in Example 2, except that Jurkat cells
expressing NFAT-driven Luciferase were used and luciferase activity
was monitored. NFAT-driven Luciferase CD3 Jurkat reporter cells
were co-cultured with SHP-77 (DLL3-positive) target cells in the
presence of monovalent and bivalent constructs containing
antigen-binding domains against the DLL3 antigen (see FIG. 4A).
Specifically, as shown in FIG. 17C, the exemplary bivalent
construct cx5352 induced substantially greater luciferase activity
in this assay compared to the exemplary monovalent constructs
cx5800 and cx5801. These results are consistent with results
observed with B7H3-targeted constructs, thereby indicating that the
activity of the constructs is not specific to a particular target
antigen.
[0625] C. Cytotoxicity
[0626] Cytotoxicity of cx5499, a DLL3-targeted CD3 constrained
binding construct formatted with two distinct sdAb binding domains
located at its amino and carboxy termini, was assessed against a
DLL3 expressing cell line, SHP-77, using an assay substantially as
described in Example 4. As shown in FIG. 18A, cx5499 induced potent
T-cell mediated cytotoxicity directed toward the SHP-77 cell
line.
[0627] D. T Cell Modulation
[0628] To further assess T cell modulation, exemplary multispecific
CD3 constrained binding constructs were assessed by monitoring the
ability of the constructs to modulate T cell activation markers. To
assess T cell activation, suspension cells from T cell cytotoxicity
assays above, involving culture of T cells with DLL3 positive
SHP-77 cells in the presence of cx5499, in the presence of an
exemplary DLL3-targeted constrained CD3 engaging constructs, were
collected. Cells were stained with a live/dead stain and
fluorophore-conjugated anti-CD4, anti-CD8, anti-CD25 and/or
anti-CD69 antibodies. Cells were analyzed using a SONY SA3800
spectral analyzer and CD4+ or CD8+ T cell activation was determined
by measuring expression levels of CD25 or CD69 or percent CD25- or
CD69-positive.
[0629] FIG. 18B and FIG. 18D depict results for CD25 expression on
CD4 T cells or CD8 T cells, respectively, upon culture of T cells
with DLL3 positive, SHP-77 cells, in the presence of an exemplary
DLL3-targeted constrained CD3 engaging construct, cx5499. FIG. 18C
and FIG. 18E depict results for CD69 expression on CD4 cells or CD8
T cells, respectively, upon culture of T cells with DLL3 positive,
SHP-77 cells, in the presence of an exemplary DLL3-targeted
constrained CD3 engaging construct, cx5499. The results showed that
cx5499 mediated a dose-dependent DLL3-dependent T-cell activation
via CD3 binding, as evidenced by increased expression of CD25 and
CD69 on CD4+ and CD8+ T cells.
[0630] E. Summary
[0631] Together, these results demonstrate that constrained
anti-CD3 constructs formatted with anti-DLL3 sdAb binding domains
are capable of binding to a DLL3-expressing cell line, SHP-77, and
eliciting antigen-dependent T-cell cytotoxicity and activation.
This result is consistent with a finding that the constrained CD3
engaging constructs of the disclosure have broad applicability to
specifically target numerous tumor antigens and elicit T-cell
cytotoxicity and activation against target-expressing cells.
[0632] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the disclosure,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
TABLE-US-00011 SEQUENCE TABLE SEQ ID DESCRIPTION NO SEQUENCE 1
PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV IgG1 Fc
DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA
KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD
SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 2 PAPGGPSVFL
FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV xELL Fc EVHNAKTKPR
EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP
PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK 3 PAPPVAGPSV FLFPPKPKDT
LMISRTPEVT CVVVDVSHED PEVQFNWYVD IgG2 Fc GVEVHNAKTK PREEQFNSTF
RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA PIEKTISKTK GQPREPQVYT LPPSREEMTK
NQVSLTCLVK GFYPSDISVE WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG
NVFSCSVMHE ALHNHYTQKS LSLSPGK 4 PAPELLGGPS VFLFPPKPKD TLMISRTPEV
TCVVVDVSHE DPEVQFKWYV IgG3 Fc DGVEVHNAKT KPREEQYNST FRVVSVLTVL
HQDWLNGKEY KCKVSNKALP APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV
KGFYPSDIAV EWESSGQPEN NYNTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNIFSCSVMH
EALHNRFTQK SLSLSPGK 5 PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE
DPEVQFNWYV IgG4 Fc DGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY
KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV
EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK
SLSLSLGK 6 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV
IgG4 Fc DGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP
SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN
NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 7
EPKSSDKTHTCPPC Hinge 8 DKTHTCPPC Hinge 9 ESKYGPPCPPC Hinge 10
GGSGGS (GGS).sub.2 11 GGSGGSGGS (GGS).sub.3 12 GGSGGSGGSGGS
(GGS).sub.4 13 GGSGGSGGSGGSGGS (GGS).sub.5 14
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH
RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR
HGNFGNSYVSWFAYWGQGTLVTVSA 15
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIG anti-CD3 VL
GTNKRAPGVPARFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNLWVFGG GTKLTVL 16
TYAMN anti-CD3 VH CDR1 17 RIRSKYNNYATYYADSVKD anti-CD3 VH CDR2 18
HGNFGNSYVSWFAY anti-CD3 VH CDR3 19 RSSTGAVTTSNYAN anti-CD3 VL CDR1
20 GTNKRAP anti-CD3 VL CDR2 21 ALWYSNLWV anti-CD3 VL CDR3 22 LEAD
Granzyme B substrate 23 RQAR Granzyme B substrate 24 PAGL MMP
substrate 25 TGLEADGSPAGLGRQARVG Linker 26 TGLEADGSRQARVGPAGLG
Linker 27 TGSPAGLEADGSRQARVGS Linker 28 TGPAGLGLEADGSRQARVG Linker
29 TGRQARVGLEADGSPAGLG Linker 30 TGSRQARVGPAGLEADGS Linker 31
TGPAGLGSRQARVGLEADGS Linker 32
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR anti-CD3 VH1
IRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 33
EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH2
RIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVR
HGNFGNSYVSWFAYWGQGTLVTVSS 34
EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR anti-CD3 VH3
IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNNLKIEDTAMYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 35
EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR anti-CD3 VH4
IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKIEDTAMYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 36
EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR anti-CD3 VHS
IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKIEDTAMYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 37
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRI anti-CD3 VH6
RSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 38
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVGR anti-CD3 VH7
IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH
GNFGDSYVSWFAYWGQGTLVTVSS 39
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVA anti-CD3 VH8
RIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCV
RHGNFGNSYISYWAYWGQGTLVTVS 40
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH9
RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR
HGNFGNSYVSWFAYWGQGTTVTVSS 41
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH10
RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR
HGNFGNSYVSYFAYWGQGTTVTVSS 42
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH11
RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR
HGNFGNSYVSWFAYWGQGTLVTVSS 43
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH12
RIRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVR
HGNFGNSYVSWFAYWGQGTLVTVKP 44
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH13
IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVKP 45
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH14
RIRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVR
HGNFGNSYVSWFAYWGCGTLVTVKP 46
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVAR anti-CD3 VH15
IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 47
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVSR anti-CD3 VH16
IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 48
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSR anti-CD3 VH17
IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 49
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVAR anti-CD3 VH18
IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 50
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVSR anti-CD3 VH19
IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 51
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVSRI anti-CD3 VH20
RSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 52
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVGR anti-CD3 VH21
IRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 53
EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH22
IRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVR
HGNFGNSYVSWFAYWGQGTLVTVSS 54
EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH23
IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNNLKIEDTAMYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 55
EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH24
IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKIEDTAMYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 56
EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH25
IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKIEDTAMYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 57
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVSRI anti-CD3 VH26
RSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTLVTVSS 58
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKCLEWVGRI anti-CD3 VH27
RSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH
GNFGDSYVSWFAYWGQGTLVTVSS 59
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKCLEWVA anti-CD3 VH28
RIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCV
RHGNFGNSYISYWAYWGQGTLVTVS 60
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH29
IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH
GNFGNSYVSWFAYWGQGTTVTVSS 61
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH30
IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH
GNFGNSYVSYFAYWGQGTTVTVSS 62
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKCLEWVA anti-CD3 VH31
RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR
HGNFGNSYVSWFAYWGQGTLVTVSS 63
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIG anti-CD3 VL1
GTNKRAPGVPARFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNLWVFGG GTKLTVL 64
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIG anti-CD3 VL2
GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG CGTKLEIK 65
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG anti-CD3 VL3
GTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFG GGTKLTVL 66
QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL4
GTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVFGG GTKLTVL 67
QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL5
GTNKRAPGVPARFSGSILGNKAALTITGAQADDESIYFCALWYSNLWVFGG GTKLTVL 68
QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL6
GTNKRAPGVPARFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFG GGTKLTVL
69 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIG anti-CD3 VL7
GTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFG GGTKLTVL 70
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIG anti-CD3 VL8
GTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVL 71
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAFRGLIG anti-CD3 VL9
GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG GGTKLEIK 72
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAFRGLIG anti-CD3 VL10
GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG CGTKLEIK 73
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQCFRGLIG anti-CD3 VL11
GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG EGTKLEIK 74
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIG anti-CD3 VL12
GTNKRAPGVPARFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNLWVFGC GTKLTVL 75
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIG anti-CD3 VL13
GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG GGTKLEIK 76
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG anti-CD3 VL14
GTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFG CGTKLTVL 77
QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL15
GTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVFGG GTKLTVL 78
QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL16
GTNKRAPGVPARFSGSILGNKAALTITGAQADDESIYFCALWYSNLWVFGC GTKLTVL 79
QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL17
GTNKRAPGVPARFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFG CGTKLTVL 80
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIG anti-CD3 VL18
GTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFG CGTKLTVL 81
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIG anti-CD3 VL19
GTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFG CGTKLTVL 82
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Knob Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPT 83
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPE Hole Fc
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPT 84
DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Knob Fc
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPT 85
DKTHTCPPCPAPGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKF Hole Fc
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPT 86
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Knob Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPG 87
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPE Hole Fc
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPG 88
DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Knob Fc
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPG 89
DKTHTCPPCPAPGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKF Hole Fc
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPG 90
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Hole Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS
VMHEALHNRYTQKSLSLSPT 91
DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Hole Fc
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV
MHEALHNRYTQKSLSLSPT 92
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Hole Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS
VMHEALHNRYTQKSLSLSPG 93
DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Hole Fc
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV
MHEALHNRYTQKSLSLSPG 94
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Knob Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVVHEALHNHYTQKSLSLSPT 95
DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Knob Fc
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVV
HEALHNHYTQKSLSLSPT 96
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Knob Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVVHEALHNHYTQKSLSLSPG 97
DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Knob Fc
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVV
HEALHNHYTQKSLSLSPG 98
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Hole Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS
VVHEALHNRYTQKSLSLSPT 99
DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Hole Fc
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVH
EALHNRYTQKSLSLSPT 100
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Hole Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS
VVHEALHNRYTQKSLSLSPG 101
DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Hole Fc
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVH
EALHNRYTQKSLSLSPG 102 PGGGG Peptide Linker 103 GGGG Peptide Linker
104 GPAGLGLEPDGSRQARVG Linker 105 GGSGGGGIEPDIGGSGGS Linker 106
GGSGGGGLEADTGGSGGS Linker 107 GSIEPDIGS Linker 108 GSLEADTGS Linker
109 GGSGGGGIEPDGGGSGGS Linker 110 GGSGGGGIEPDVGGSGGS Linker 111
GGSGGGGIEPDSGGSGGS Linker 112 GGSGGGGIEPDTGGSGGS Linker 113
GGGSLEPDGSGS Linker 114 GPAGLGLEADGSRQARVG Linker 115
GGEGGGGSGGSGGGS Linker 116 GSSAGSEAGGSGQAGVGS Linker 117
GGSGGGGLEAEGSGGGGS Linker 118 GGSGGGGIEPDPGGSGGS Linker 119
GGGGGSGGGGGSGGGGGS Linker 120
QLQLQESGGGLVQPGGSLRLSCAASGFTLDNYAIGWFRQAPGKEREGVSCIS FR alpha sdAb
SSDGSTYYADSVKGRFTISRNNAKGTVYLLMNSLKPEDTAVYYCATELVPA
CTYSNGRGPLDGMDYWGKGTQVTVKP 121
EVQLLESGGGEVQPGGSLRLSCAASGSIFSIDATAWYRQAPGKQRELVAIITS FR alpha sdAb
SGSTNYPESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCNAITRYGGS TYDFWGQGTLVTVKP
122 EVQPGGSLRLSCAASETFGVVFTLGWYRQAPGKGREFVARVTGTDTVDYA FR alpha
sdAb ESVKGRFTISSDFARNTVYLQMNSLRAEDTAVYYCNTGAYWGQGTLVTVK P 123
EVQLVESGGGLVQPGGSLRLSCAASGFILDYYAIGWFRQAPGKEREGVLCID cMET sdAb
ASDDITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCATPIGLSS
SCLLEYDYDYWGQGTLVTVKP 124
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAY B7H3 scFv
ISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGREN
IYYGSRLDYWGQGTTVTVSSSGGGGSGGGGSGGGGSDIQLTQSPSFLSASVG
DRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGS
GTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK 125
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMG CD20 scFv
RIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNV
FDGYWLVYWGQGTLVTVSGSGGGGSGGGGTGGGGSDIVMTQTPLSLPVTP
GEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFS
GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK 126
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYVY DLL3 scFv
YSGTTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFY
FDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERVTLSC
RASQRVNNNYLAWYQQRPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLT
ISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIK 127
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAY B7H3 Fd
ISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGREN
IYYGSRLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSC 128
DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSAS B7H3 LC
YRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLE
IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 129
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA 5T4 Fd
RIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCV
RQWDYDVRAMNYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSC 130
DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWA 5T4 LC
STRLTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTK
LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ
SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 131
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPGKGLEWIGY gpNMB Fd
IYYSGSTYSNPSLKSRVTISVDTSKNQFSLTLSSVTAADTAVYYCARGYNWN
YFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSC
132 EIVMTQSPATLSVSPGERATLSCRASQSVDNNLVWYQQKPGQAPRLLIYGAS gpNMB LC
TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPWTFGQGTK
VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ
SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 133
QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMA DLL3 Fd
WINTYTGEPTYADDFKGRFAFSLETSASTASLQIINLKNEDTATYFCARIGDS
SPSDYWGQGTTLTVSSSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSC 134
SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVVWYQQKPGQSPKLLIYYAS DLL3 LC
NRYTGVPDRFAGSGYGTDFSFTISTVQAEDLAVYFCQQDYTSPWTFGGGTK
LEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ
SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 135
GGGGGS Peptide Linker 136 IEPDI Linker 137 LEADT Linker 138 IEPDG
Linker 139 IEPDV Linker 140 IEPDS Linker 141 IEPDT Linker 142 LEPD
Linker 143 LEAE Linker 144 IEPDP Linker 145
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG Second
GTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFG Polypeptide
GGTKLTVLGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGM Chain of B7-H3
HWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNS x CD3
Bispecific LRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGGCGGGKVAALKE
DART-A KVAALKEKVAALKEKVAALKE Diabody 146
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE Third
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC Polypeptide
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFY Chain of B7-H3
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS x CD3
Bispecific CSVMHEALHNRYTQKSLSLSPGK DART-A Diabody 147
GGSGGGGSGGGGSGGGGS Linker 148 TGGSGGGGIEPDIGGSGGS Linker 149 GGGGS
Linker 150 X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 (P4 P3 P2 P1
.dwnarw. P1') Linker consensus X1 = I, L, Y, M, F, V, or A; (P4 =
I, L, Y, M, F, V, or A) X2 = A, G, S, V, E, D, Q, N, or Y; (P3 = A,
G, S, V, E, D, Q, N, or Y) X3 = H, P, A, V, G, S, or T; (P2 = H, P,
A, V, G, S, or T) X4 = D or E; (P1 = D or E) X5 = I, L, Y, M, F, V,
T, S, G or A (P1' = I, L, Y, M, F, V, T, S, G or A) 151 X1 E X3 D
X5 (P4 P3 P2 P1 .dwnarw. P1') Linker consensus X1 = I or L; (P4 = I
or L) (P3 = E) X3 = P or A; (P2 = P or A) X5 = I, V, T, S, or G
(P1' = I, V, T, S, or G) 152 LEPDG Linker 153 LEADG Linker 154
X.sub.1QARX.sub.5 (P1QAR.dwnarw.(A/V)) Linker consensus X1 = any
amino acid; (P1 is any amino acid) X5 = A or V 155 RQARX.sub.5
(RQAR(A/V)) Linker consensus X5 = A or V 156 RQARV Linker 157 X1X2
X3 X4 (P3 P2 P1 .dwnarw. P1') Linker consensus X1 = P, V or A; (P3
= P, V or A) X2 = Q or D; (P2 = Q or D) X3 = A or N; (P1 = A or N)
X4 = L, I or M (P1' = L, I or M) 158 PX2X3X4 (P3P2 P1 .dwnarw. P1')
Linker consensus (P3 = P) X2 = Q or D; (P2 = Q or D) X3 = A or N;
(P1 = A or N) X4 = L or I (P1' = L or I) 159 GSGATNFSLLKQAGDVEENPGP
P2A 160 ATNFSLLKQAGDVEENPGP P2A 161 QCTNYALLKLAGDVESNPGP E2A 162
VKQTLNFDLLKLAGDVESNPGP F2A 163 EGRGSLLTCGDVEENPGP T2A 164
LEGGGEGRGSLLTCGDVEENPGPR T2A 165
GGATCTGGAGCAACAAACTTCTCACTACTCAAACAAGCAGGTGACGTGG AGGAGAATCCCGGACCC
P2A DNA 166 GSPAGLEADGSRQARVGS Linker 167 EVQLVESGGGL VQPKGSLKLS
CAASGFTFNT YAMNWVRQAP anti-5T4 VH GKGLEWVARI RSKSNNYATY YADSVKDRFT
ISRDDSQSML YLQMNNLKTE DTAMYXCVRQ WDYDVRAMNY WGQGTSVTVS S 168
DIVMTQSHIF MSTSVGDRVS ITCKASQDVD anti-5T4 VL TAVAWYQQKP GQSPKLLIYW
ASTRLTGVPD RFTGSGSGTD FTLTISNVQS EDLADYFCQQ YSSYPYTFGG GTKLEIK 169
DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKALIYS First
Polypeptide ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNNYPFTFGQ
Chain of B7-H3 GTKLEIKGGG SGGGGEVQLV ESGGGLVQPG GSLRLSCAAS x CD3
Bispecific GFTFSTYAMN DART-A WVRQAPGKGL EWVGRIRSKY NNYATYYADS
VKDRFTISRD Diabody DSKNSLYLQM NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW
GQGTLVTVSS GGCGGGEVAA LEKEVAALEK EVAALEKEVA ALEKGGGDKT HTCPPCPAPE
AAGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE
EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP
SREEMTKNQV SLWCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD
KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK 170 GGGGSGGGGSGGGGS Linker
171 GGS(GGS)n Linker wherein n is 0 to 10 172 (GGGGGS)n Linker
wherein n is 1 to 4 173 (GGGGS)n Linker wherein n is 1 to 10 174
Gly.sub.xXaa-Gly.sub.y-Xaa-Gly.sub.z Linker Xaa is independently
selected from A, V, L, I, M, F, W, P, G, S, T, C, Y, N, Q, K, R, H,
D, or E x, y, and z are each integers in the range from 1-5 175
Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly Linker Xaa is
independently selected from A, V, L, I, M, F, W, P, G, S, T, C, Y,
N, Q, K, R, H, D, or E 176 ATTTGSSPGPT Linker 177 GGGGG-C-GGGGG
Linker 178 (EAAAK)n Linker n = 2-20 179 AS-(AP)n-GT Linker n = 2-20
180 AS-(EAAAK)n-GT Linker n = 2-20 181 (GGGGA)n Linker n = 2-20 182
(PGGGS)n Linker n = 2-20 183 (AGGGS)n Linker n = 2-20 184
GGS-(EGKSSGSGSESKST)n-GGS Linker n = 2-20 185 (SSSSG)n Linker n =
1-9 186 SSSASASSA Linker 187 GSPGSPG Linker 188 QVQLQESGPG
LVKPSETLSL TCTVSGGSIS SYYWSWIRQP PGKGLEWIGY DLL3 scFv VYYSGTTNYN
PSLKSRVTIS VDTSKNQFSL KLSSVTAADT AVYYCASIAV TGFYFDYWGQ GTLVTVSSGG
GGSGGGGSGG GGSEIVLTQS PGTLSLSPGE RVTLSCRASQ RVNNNYLAWY QQRPGQAPRL
LIYGASSRAT GIPDRFSGSG SGTDFTLTIS RLEPEDFAVY YCQQYDRSPL TFGGGTKLEI K
189 QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA CD20 VH PGQGLEWMGR
IFPGDGDTDY NGKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARNV FDGYWLVYWG
QGTLVTVSS 190 DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW
YLQKPGQSPQ CD20 VL LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV
YYCAQNLELP YTFGGGTKVE IKRTV 191
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAFRGLIG anti-CD3 VL35
GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG (CON) CGTKLTVL
192 GGGGG linker 193 GGGGSGGGGSGGGGS linker 194
DKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEV Fc-Het-1
KFNWYVDGVEVHNAKTKPREEEYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCDVSGFYPS
DIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWEQGDVFSCS
VMHEALHNHYTQKSLSLSPGK 195
DKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEV Fc-Het-2
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREQMTKNQVKLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK 196
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR CD3-VH32
IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH
GNFGDSYVSWFAYWGQGTLVTVSS 197
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVGR CD3-VH33
IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH
GNFGDSYVSWFAYWGQGTLVTVSS 198
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVAR CD3-VH34
IRSKYNNYATYYADTVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVR
HGNFGDSYVSWFAYWGQGTLVTV 199
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIG CD3-VL20
GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG GGTKLTVL 200
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIG CD3-VL21
GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG CGTKLTVL 201
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Knob Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSP 202
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPE Hole Fc
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSP 203
DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Knob Fc
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSP 204
DKTHTCPPCPAPGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKF Hole Fc
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSP 205
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Hole Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS
VMHEALHNRYTQKSLSLSP 206
DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Hole Fc
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV
MHEALHNRYTQKSLSLSP 207
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Knob Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVVHEALHNHYTQKSLSLSP 208
DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Knob Fc
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVV
HEALHNHYTQKSLSLSP 209
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Hole Fc
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS
VVHEALHNRYTQKSLSLSP 210
DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Hole Fc
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVH
EALHNRYTQKSLSLSP 211 GFTFNTYAMN anti-CD3 VH CDR1 212 RIRSKYNNYATY
anti-CD3 VH CDR2 213 QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA
CD20 scFv PGQGLEWMGR IFPGDGDTDY NGKFKGRVTI TADKSTSTAY MELSSLRSED
TAVYYCARNV FDGYWLVYWG QGTLVTVSSG GGGSGGGGSG GGGSDIVMTQ TPLSLPVTPG
EPASISCRSS KSLLHSNGIT YLYWYLQKPG QSPQLLIYQM SNLVSGVPDR FSGSGSGTDF
TLKISRVEAE DVGVYYCAQN LELPYTFGGG TKVEIK 214
EVQLVESGGGEVQPGGSLRLSCAASGFSFSSNVMMWVRQAPGKGLEWVSTIYSSG B7H3 sdAb
TGTFYAESVKGRFTISRDNAKNTLYLQMSSLRPEDTAVYYCATSGPVRGWGPRSQGT B7h3
hz1A5v51 LVTVKP 215
EVQLVESGGGEVQPGGSLRLSCAASGSTFSSYHMSWFRQAPGKQREPVATS sdAb B7H3
HHGGTTNYAGSVKGRFTISRDNAKNTVYLQMNTLRAEDTAVYYCKADHG hz58E05v27
YQGRGYWGQGTLVTVKP 216
EVQLVESGGGEVQPGGSLRLSCAASGFTFSSYHMSWFRQAPGKQRELVATSHHGGT sdAb B7H3
TNYAGSVKGRFTISRDNAKNTVYLQMNTLRAEDTAVYYCKADHGYQGRGYWGQGT hz58E05v55
LVTVKP 217 EVQLVESGGGEVQPGGSLRLSCAASGFTFSSYHMSWFRQAPGKQREPVATS sdAb
B7H3 HHGGTTNYAGSVKGRFTISRDNAKNTVYLQMNTLRAEDTAVYYCKADHG hz58E05v48
YQGRGYWGQGTLVTVKP 218
EVQLVESGGGEVQPGGSLRLSCAPSERTFSTYTMGWFRQAPGKEREFVAVV sdAb B7H3
NWGGGSKYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAAGG hz57B04v24
AYSGPYYDTRQYTYWGQGTLVTVKPGG 219
EVQLVESGGGEVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAGF sdAb DLL3
TGDTNTIYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAADVQLF hz10D9v7
SRDYEFYWGQGTLVTVKP 220
EVQLVESGGGEVQPGGSLRLSCGPSEIITSDKSMGWVRQAPGKQRNLVAGIS sdAb DLL3
NVGSTNYAQSVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYARDFEN hz8E7v16
EYWGQGTLVTVKP 221 GFSFSINAMG 41BB CDR1 222 AIESGRNTV 41BB CDR2 223
LKGNRVVSPSVAY 41BB CDR3 224 HGNFGDSYVSWFAY CD3-VH7, VH33 CDR3 225
ALWYSNHWV CD3-VL2, VL21 CDR3 226 VLWYSNRWV CD3-VL8 CDR3 227
GFTFSTYAMN CD3 VH33 CDR1 228 RIRSKYNNYATY CD3 VH33 CDR1 229
GSSTGAVTTSNYAN CD3 VL21 CDR1 230 GTNKRAP CD3 VL21 CDR2
Sequence CWU 1
1
2301218PRTArtificial SequenceIgG1 Fc 1Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 20 25 30Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 35 40 45Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 50 55 60Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu65 70 75 80His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 85 90
95Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu 115 120 125Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 130 135 140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 165 170 175Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 180 185 190Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 195 200 205Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 2152215PRTArtificial
SequencexELL Fc 2Pro Ala Pro Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys1 5 10 15Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 20 25 30Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp 35 40 45Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 50 55 60Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp65 70 75 80Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 85 90 95Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 115 120 125Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135
140Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys145 150 155 160Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 165 170 175Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser 180 185 190Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser 195 200 205Leu Ser Leu Ser Pro Gly
Lys 210 2153217PRTArtificial SequenceIgG2 Fc 3Pro Ala Pro Pro Val
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln
Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ser Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Lys Thr Thr Pro Pro
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200
205Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 2154218PRTArtificial
SequenceIgG3 Fc 4Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 20 25 30Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp 35 40 45Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 50 55 60Glu Gln Tyr Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Leu65 70 75 80His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 85 90 95Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 100 105 110Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 115 120 125Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 130 135
140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu
Asn145 150 155 160Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp
Gly Ser Phe Phe 165 170 175Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 180 185 190Ile Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn Arg Phe Thr 195 200 205Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 210 2155218PRTArtificial SequenceIgG4 Fc 5Pro Ala
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro1 5 10 15Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 20 25
30Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
35 40 45Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 50 55 60Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu65 70 75 80His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 85 90 95Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu 115 120 125Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 130 135 140Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 165 170
175Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
180 185 190Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 195 200 205Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210
2156218PRTArtificial SequenceIgG4 Fc 6Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 20 25 30Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 35 40 45Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 50 55 60Glu Gln Phe
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu65 70 75 80His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 85 90
95Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu 115 120 125Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 130 135 140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 165 170 175Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 180 185 190Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 195 200 205Gln
Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215714PRTArtificial
SequenceHinge 7Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
Cys1 5 1089PRTArtificial SequenceHinge 8Asp Lys Thr His Thr Cys Pro
Pro Cys1 5911PRTArtificial SequenceHinge 9Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys1 5 10106PRTArtificial SequenceSynthetic -
Linker(GGS)2 10Gly Gly Ser Gly Gly Ser1 5119PRTArtificial
SequenceSynthetic - Linker (GGS)3 11Gly Gly Ser Gly Gly Ser Gly Gly
Ser1 51212PRTArtificial SequenceSynthetic - Linker (GGS)4 12Gly Gly
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser1 5 101315PRTArtificial
SequenceSynthetic - Linker (GGS)5 13Gly Gly Ser Gly Gly Ser Gly Gly
Ser Gly Gly Ser Gly Gly Ser1 5 10 1514125PRTArtificial
SequenceSynthetic - anti-CD3 VH 14Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Lys Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser
Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp
Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile65 70 75 80Leu Tyr
Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95Tyr
Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105
110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120
12515109PRTArtificial Sequenceanti-CD3 VL 15Gln Ala Val Val Thr Gln
Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu1 5 10 15Thr Val Thr Leu Thr
Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn
Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45Leu Ile Gly
Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser Gly
Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75
80Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105165PRTArtificial Sequenceanti-CD3 VH CDR1 16Thr Tyr Ala Met Asn1
51719PRTArtificial Sequenceanti-CD3 VH CDR2 17Arg Ile Arg Ser Lys
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser1 5 10 15Val Lys
Asp1814PRTArtificial Sequenceanti-CD3 VH CDR3 18His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Trp Phe Ala Tyr1 5 101914PRTArtificial
Sequenceanti-CD3 VL CDR1 19Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
Asn Tyr Ala Asn1 5 10207PRTArtificial Sequenceanti-CD3 VL CDR2
20Gly Thr Asn Lys Arg Ala Pro1 5219PRTArtificial Sequenceanti-CD3
VL CDR3 21Ala Leu Trp Tyr Ser Asn Leu Trp Val1 5224PRTArtificial
SequenceGranzyme B substrate 22Leu Glu Ala Asp1234PRTArtificial
SequenceGranzyme B substrate 23Arg Gln Ala Arg1244PRTArtificial
SequenceMMP substrate 24Pro Ala Gly Leu12519PRTArtificial
SequenceLinker 25Thr Gly Leu Glu Ala Asp Gly Ser Pro Ala Gly Leu
Gly Arg Gln Ala1 5 10 15Arg Val Gly2619PRTArtificial SequenceLinker
26Thr Gly Leu Glu Ala Asp Gly Ser Arg Gln Ala Arg Val Gly Pro Ala1
5 10 15Gly Leu Gly2719PRTArtificial SequenceLinker 27Thr Gly Ser
Pro Ala Gly Leu Glu Ala Asp Gly Ser Arg Gln Ala Arg1 5 10 15Val Gly
Ser2819PRTArtificial SequenceLinker 28Thr Gly Pro Ala Gly Leu Gly
Leu Glu Ala Asp Gly Ser Arg Gln Ala1 5 10 15Arg Val
Gly2919PRTArtificial SequenceLinker 29Thr Gly Arg Gln Ala Arg Val
Gly Leu Glu Ala Asp Gly Ser Pro Ala1 5 10 15Gly Leu
Gly3018PRTArtificial SequenceLinker 30Thr Gly Ser Arg Gln Ala Arg
Val Gly Pro Ala Gly Leu Glu Ala Asp1 5 10 15Gly
Ser3120PRTArtificial SequenceLinker 31Thr Gly Pro Ala Gly Leu Gly
Ser Arg Gln Ala Arg Val Gly Leu Glu1 5 10 15Ala Asp Gly Ser
2032125PRTArtificial Sequenceanti-CD3 VH1 32Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12533125PRTArtificial Sequenceanti-CD3 VH2 33Glu Val Lys
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
Ser65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 12534125PRTArtificial Sequenceanti-CD3 VH3
34Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ile65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr
Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 12535125PRTArtificial
Sequenceanti-CD3 VH4 35Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Ser Ile65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90
95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 12536125PRTArtificial Sequenceanti-CD3 VH5 36Glu Val Lys Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile65
70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Met
Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 12537125PRTArtificial Sequenceanti-CD3 VH6 37Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25
30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 12538125PRTArtificial Sequenceanti-CD3 VH7
38Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asp Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 12539124PRTArtificial
Sequenceanti-CD3 VH8 39Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Lys Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Ala Tyr Leu Gln Met
Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100 105 110Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 12040125PRTArtificial
Sequenceanti-CD3 VH9 40Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
12541125PRTArtificial Sequenceanti-CD3 VH10 41Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Tyr
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 12542125PRTArtificial Sequenceanti-CD3 VH11 42Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser
Ile65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 12543125PRTArtificial Sequenceanti-CD3 VH12
43Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ala Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Lys Pro 115 120 12544125PRTArtificial
Sequenceanti-CD3 VH13 44Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Cys Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ala Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met
Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro 115 120
12545125PRTArtificial Sequenceanti-CD3 VH14 45Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr65 70 75
80Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Cys Gly Thr Leu Val Thr Val Lys Pro
115 120 12546125PRTArtificial Sequenceanti-CD3 VH15 46Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn
Thr65 70 75 80Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 12547125PRTArtificial Sequenceanti-CD3 VH16
47Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ala Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 12548125PRTArtificial
Sequenceanti-CD3 VH17 48Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ala Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met
Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
12549125PRTArtificial Sequenceanti-CD3 VH18 49Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45Ala Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr65 70 75
80Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12550125PRTArtificial Sequenceanti-CD3 VH19 50Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40
45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn
Thr65 70 75 80Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 12551125PRTArtificial Sequenceanti-CD3 VH20
51Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu
Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ala Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 12552125PRTArtificial
Sequenceanti-CD3 VH21 52Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Cys Leu Glu Trp Val 35 40 45Gly Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
12553125PRTArtificial Sequenceanti-CD3 VH22 53Glu Val Lys Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45Ala Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser
Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75
80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12554125PRTArtificial Sequenceanti-CD3 VH23 54Glu Val Lys
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40
45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
Ile65 70
75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met
Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 12555125PRTArtificial Sequenceanti-CD3 VH24 55Glu Val
Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25
30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val
35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Ser Ile65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 12556125PRTArtificial Sequenceanti-CD3 VH25
56Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu
Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ile65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr
Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 12557125PRTArtificial
Sequenceanti-CD3 VH26 57Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Cys Leu Glu Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
12558125PRTArtificial Sequenceanti-CD3 VH27 58Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45Gly Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asp Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12559124PRTArtificial Sequenceanti-CD3 VH28 59Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40
45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Thr65 70 75 80Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Ile Ser Tyr Trp 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser 115 12060125PRTArtificial Sequenceanti-CD3 VH29 60Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25
30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val
35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120 12561125PRTArtificial Sequenceanti-CD3 VH30
61Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu
Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Tyr Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120 12562125PRTArtificial
Sequenceanti-CD3 VH31 62Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Lys Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Cys Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr
Ile Ser Arg Asp Asp Ser Gln Ser Ile65 70 75 80Leu Tyr Leu Gln Met
Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
12563109PRTArtificial Sequenceanti-CD3 VL1 63Gln Ala Val Val Thr
Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu1 5 10 15Thr Val Thr Leu
Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75
80Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10564109PRTArtificial Sequenceanti-CD3 VL2 64Gln Ala Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Gln Lys Pro Gly Lys Ser Pro Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly Ala65 70 75
80Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95His Trp Val Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys 100
10565109PRTArtificial Sequenceanti-CD3 VL3 65Gln Ala Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75
80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10566109PRTArtificial Sequenceanti-CD3 VL4 66Gln Ala Val Val Thr
Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75
80Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10567109PRTArtificial Sequenceanti-CD3 VL5 67Gln Ala Val Val Thr
Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser
Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75
80Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10568109PRTArtificial Sequenceanti-CD3 VL6 68Gln Ala Val Val Thr
Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser
Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75
80Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10569109PRTArtificial Sequenceanti-CD3 VL7 69Gln Ala Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65 70 75
80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10570109PRTArtificial Sequenceanti-CD3 VL8 70Gln Thr Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30Asn Tyr Pro
Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45Leu Ile
Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val65 70 75
80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn
85 90 95Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10571109PRTArtificial Sequenceanti-CD3 VL9 71Gln Ala Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly Ala65 70 75
80Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95His Trp Val Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10572109PRTArtificial Sequenceanti-CD3 VL10 72Gln Ala Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly Ala65 70 75
80Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95His Trp Val Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys 100
10573109PRTArtificial Sequenceanti-CD3 VL11 73Gln Ala Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Gln Lys Pro Gly Gln Cys Phe Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly Ala65 70 75
80Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95His Trp Val Phe Gly Glu Gly Thr Lys Leu Glu Ile Lys 100
10574109PRTArtificial Sequenceanti-CD3 VL12 74Gln Ala Val Val Thr
Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu1 5 10 15Thr Val Thr Leu
Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75
80Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu 100
10575109PRTArtificial Sequenceanti-CD3 VL13 75Gln Ala Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ser Pro
Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr
Ile Ser Gly Ala65 70 75 80Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys
Ala Leu Trp Tyr Ser Asn 85 90 95His Trp Val Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 10576109PRTArtificial Sequenceanti-CD3 VL14
76Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro
Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr
Ile Thr Gly Ala65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Cys Gly Thr Lys
Leu Thr Val Leu 100 10577109PRTArtificial Sequenceanti-CD3 VL15
77Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe
Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
Ile Thr Gly Ala65 70 75 80Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 10578109PRTArtificial Sequenceanti-CD3 VL16
78Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe
Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
Ala Arg Phe 50 55 60Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr
Ile Thr Gly Ala65 70 75 80Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Cys Gly Thr Lys
Leu Thr Val Leu 100 10579109PRTArtificial Sequenceanti-CD3 VL17
79Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe
Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
Ala Arg Phe 50 55 60Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr
Ile Thr Gly Ala65 70 75 80Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Cys Gly Thr Lys
Leu Thr Val Leu 100 10580109PRTArtificial Sequenceanti-CD3 VL18
80Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe
Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr
Leu Ser Gly Ala65 70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Cys Gly Thr Lys
Leu Thr Val Leu 100 10581109PRTArtificial Sequenceanti-CD3 VL19
81Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser
Gly 20 25 30Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro
Arg Gly 35 40 45Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr
Leu Ser Gly Val65 70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys
Val Leu Trp Tyr Ser Asn 85 90 95Arg Trp Val Phe Gly Cys Gly Thr Lys
Leu Thr Val Leu 100 10582226PRTArtificial SequenceKnob Fc 82Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Cys Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Trp Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220Pro Thr22583226PRTArtificial
SequenceHole Fc 83Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30Arg Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro
Thr22584223PRTArtificial SequenceKnob Fc 84Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val65 70 75
80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 115 120 125Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Trp Cys 130 135 140Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200
205Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Thr 210
215 22085223PRTArtificial SequenceHole Fc 85Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Arg Ser Arg 20 25 30Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val65 70 75
80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Cys Thr Leu 115 120 125Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Ser Cys 130 135 140Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175Ser Asp Gly Ser
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200
205Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Thr 210
215 22086226PRTArtificial SequenceKnob Fc 86Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140Leu Trp Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200
205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220Pro Gly22587226PRTArtificial SequenceHole Fc 87Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30Arg Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 115 120 125Cys Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Ser Cys Ala Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220Pro Gly22588223PRTArtificial
SequenceKnob Fc 88Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 20 25 30Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val65 70 75 80Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125Pro
Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135
140Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser145 150 155 160Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp 165 170 175Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser 180 185 190Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala 195 200 205Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 210 215 22089223PRTArtificial
SequenceHole Fc 89Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Arg Ser Arg 20 25 30Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val65 70 75 80Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser
Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 115 120 125Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140Ala
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser145 150
155 160Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp 165 170 175Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
Asp Lys Ser 180 185 190Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala 195 200 205Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 210 215 22090226PRTArtificial SequenceHole
Fc 90Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Cys Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Ser
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn Arg Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220Pro Thr22591223PRTArtificial
SequenceHole Fc 91Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 20 25 30Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val65 70 75 80Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 115 120 125Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135
140Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser145 150 155 160Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp 165 170 175Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
Leu Thr Val Asp Lys Ser 180 185 190Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala 195 200 205Leu His Asn Arg Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Thr 210 215 22092226PRTArtificial
SequenceHole Fc 92Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn
Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro
Gly22593223PRTArtificial SequenceHole Fc 93Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val65 70 75
80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Cys Thr Leu 115 120 125Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Ser Cys 130 135 140Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175Ser Asp Gly Ser
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200
205Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210
215 22094226PRTArtificial SequenceKnob Fc 94Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140Leu Trp Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val 195 200
205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220Pro Thr22595223PRTArtificial SequenceKnob Fc 95Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser1 5 10 15Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25
30Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val65 70 75 80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 115 120 125Pro Pro Cys Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135 140Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170
175Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
180 185 190Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His
Glu Ala 195 200 205Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Thr 210 215 22096226PRTArtificial SequenceKnob Fc 96Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25
30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Cys Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Trp Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Val 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220Pro Gly22597223PRTArtificial
SequenceKnob Fc 97Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Tyr Ile Ser Arg 20 25 30Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val65 70 75 80Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125Pro
Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135
140Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser145 150 155 160Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp 165 170 175Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser 180 185 190Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Val His Glu Ala 195 200 205Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 210 215 22098226PRTArtificial
SequenceHole Fc 98Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Tyr 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Val 195 200 205His Glu Ala Leu His Asn
Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro
Thr22599223PRTArtificial SequenceHole Fc 99Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val65 70 75
80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Cys Thr Leu 115 120 125Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Ser Cys 130 135 140Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175Ser Asp Gly Ser
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His Glu Ala 195 200
205Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Thr 210
215 220100226PRTArtificial SequenceHole Fc 100Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70
75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140Leu Ser Cys Ala Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val 195 200
205His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220Pro Gly225101223PRTArtificial SequenceHole Fc 101Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser1 5 10 15Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25
30Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val65 70 75 80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Cys Thr Leu 115 120 125Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140Ala Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170
175Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
180 185 190Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His
Glu Ala 195 200 205Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 210 215 2201025PRTArtificial SequencePeptide Linker
102Pro Gly Gly Gly Gly1 51034PRTArtificial SequencePeptide Linker
103Gly Gly Gly Gly110418PRTArtificial SequenceLinker 104Gly Pro Ala
Gly Leu Gly Leu Glu Pro Asp Gly Ser Arg Gln Ala Arg1 5 10 15Val
Gly10518PRTArtificial SequenceLinker 105Gly Gly Ser Gly Gly Gly Gly
Ile Glu Pro Asp Ile Gly Gly Ser Gly1 5 10 15Gly
Ser10618PRTArtificial SequenceLinker 106Gly Gly Ser Gly Gly Gly Gly
Leu Glu Ala Asp Thr Gly Gly Ser Gly1 5 10 15Gly
Ser1079PRTArtificial SequenceLinker 107Gly Ser Ile Glu Pro Asp Ile
Gly Ser1 51089PRTArtificial SequenceLinker 108Gly Ser Leu Glu Ala
Asp Thr Gly Ser1 510918PRTArtificial SequenceLinker 109Gly Gly Ser
Gly Gly Gly Gly Ile Glu Pro Asp Gly Gly Gly Ser Gly1 5 10 15Gly
Ser11018PRTArtificial SequenceLinker 110Gly Gly Ser Gly Gly Gly Gly
Ile Glu Pro Asp Val Gly Gly Ser Gly1 5 10 15Gly
Ser11118PRTArtificial SequenceLinker 111Gly Gly Ser Gly Gly Gly Gly
Ile Glu Pro Asp Ser Gly Gly Ser Gly1 5 10 15Gly
Ser11218PRTArtificial SequenceLinker 112Gly Gly Ser Gly Gly Gly Gly
Ile Glu Pro Asp Thr Gly Gly Ser Gly1 5 10 15Gly
Ser11312PRTArtificial SequenceLinker 113Gly Gly Gly Ser Leu Glu Pro
Asp Gly Ser Gly Ser1 5 1011418PRTArtificial SequenceLinker 114Gly
Pro Ala Gly Leu Gly Leu Glu Ala Asp Gly Ser Arg Gln Ala Arg1 5 10
15Val Gly11515PRTArtificial SequenceLinker 115Gly Gly Glu Gly Gly
Gly Gly Ser Gly Gly Ser Gly Gly Gly Ser1 5 10 1511618PRTArtificial
SequenceLinker 116Gly Ser Ser Ala Gly Ser Glu Ala Gly Gly Ser Gly
Gln Ala Gly Val1 5 10 15Gly Ser11718PRTArtificial SequenceLinker
117Gly Gly Ser Gly Gly Gly Gly Leu Glu Ala Glu Gly Ser Gly Gly Gly1
5 10 15Gly Ser11818PRTArtificial SequenceLinker 118Gly Gly Ser Gly
Gly Gly Gly Ile Glu Pro Asp Pro Gly Gly Ser Gly1 5 10 15Gly
Ser11918PRTArtificial SequenceLinker 119Gly Gly Gly Gly Gly Ser Gly
Gly Gly Gly Gly Ser Gly Gly Gly Gly1 5 10 15Gly
Ser120129PRTArtificial SequenceFR alpha sdAb 120Gln Leu Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asn Tyr 20 25 30Ala Ile Gly
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45Ser Cys
Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Lys Gly Thr Val Tyr65 70 75
80Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Thr Glu Leu Val Pro Ala Cys Thr Tyr Ser Asn Gly Arg Gly
Pro 100 105 110Leu Asp Gly Met Asp Tyr Trp Gly Lys Gly Thr Gln Val
Thr Val Lys 115 120 125Pro121119PRTArtificial SequenceFR alpha sdAb
121Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile
Asp 20 25 30Ala Thr Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu
Leu Val 35 40 45Ala Ile Ile Thr Ser Ser Gly Ser Thr Asn Tyr Pro Glu
Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Val Tyr Leu65 70 75 80Gln Met Ser Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Asn 85 90 95Ala Ile Thr Arg Tyr Gly Gly Ser Thr
Tyr Asp Phe Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Lys Pro
115122101PRTArtificial SequenceFR alpha sdAb 122Glu Val Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu1 5 10 15Thr Phe Gly Val
Val Phe Thr Leu Gly Trp Tyr Arg Gln Ala Pro Gly 20 25 30Lys Gly Arg
Glu Phe Val Ala Arg Val Thr Gly Thr Asp Thr Val Asp 35 40 45Tyr Ala
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Ser Asp Phe Ala 50 55 60Arg
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr65 70 75
80Ala Val Tyr Tyr Cys Asn Thr Gly Ala Tyr Trp Gly Gln Gly Thr Leu
85 90 95Val Thr Val Lys Pro 100123125PRTArtificial SequencecMET
sdAb 123Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp
Tyr Tyr 20 25 30Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Gly Val 35 40 45Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Gly Val Tyr Tyr Cys 85 90 95Ala Thr Pro Ile Gly Leu Ser Ser
Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Lys Pro 115 120 125124245PRTArtificial
SequenceB7H3 scFv 124Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Phe 20 25 30Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Tyr Ile Ser Ser Asp Ser Ser
Ala Ile Tyr Tyr Ala Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Gly Arg
Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp 100 105 110Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser 115 120
125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln
130 135 140Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr145 150 155 160Cys Lys Ala Ser Gln Asn Val Asp Thr Asn Val
Ala Trp Tyr Gln Gln 165 170 175Lys Pro Gly Lys Ala Pro Lys Ala Leu
Ile Tyr Ser Ala Ser Tyr Arg 180 185 190Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 210 215 220Tyr Cys Gln
Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr225 230 235
240Lys Leu Glu Ile Lys 245125247PRTArtificial SequenceCD20 scFv
125Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr
Ser 20 25 30Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn
Gly Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Val Phe Asp Gly Tyr Trp
Leu Val Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Thr Gly Gly Gly
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu 130 135 140Ser Leu Pro
Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser145 150 155
160Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr
165 170 175Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln
Met Ser 180 185 190Asn Leu Val Ser Gly Val Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Lys Ile Ser Arg Val
Glu Ala Glu Asp Val Gly 210 215 220Val Tyr Tyr Cys Ala Gln Asn Leu
Glu Leu Pro Tyr Thr Phe Gly Gly225 230 235 240Gly Thr Lys Val Glu
Ile Lys 245126241PRTArtificial SequenceDLL3 scFv 126Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30Tyr Trp
Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly
Tyr Val Tyr Tyr Ser Gly Thr Thr Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ser Ile Ala Val Thr Gly Phe Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr 100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu 130 135 140Ser Leu Ser Pro Gly Glu Arg Val
Thr Leu Ser Cys Arg Ala Ser Gln145 150 155 160Arg Val Asn Asn Asn
Tyr Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln 165 170 175Ala Pro Arg
Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile 180 185 190Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200
205Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
210 215 220Tyr Asp Arg Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile225 230 235 240Lys127225PRTArtificial SequenceB7H3 Fd 127Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp
Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly
Ser Arg Leu Asp Tyr Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser 210 215 220Cys225128214PRTArtificial SequenceB7H3
LC 128Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp
Thr Asn 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Ala Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Tyr Asn Asn Tyr Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
210129225PRTArtificial Sequence5T4 Fd 129Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Lys Gly1 5 10 15Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile
Arg Ser
Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp
Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met65 70 75 80Leu Tyr
Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95Tyr
Cys Val Arg Gln Trp Asp Tyr Asp Val Arg Ala Met Asn Tyr Trp 100 105
110Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215
220Cys225130214PRTArtificial Sequence5T4 LC 130Asp Ile Val Met Thr
Gln Ser His Ile Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp
Ala Ser Thr Arg Leu Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75
80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210131222PRTArtificial SequencegpNMB Fd
131Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser
Phe 20 25 30Asn Tyr Tyr Trp Ser Trp Ile Arg His His Pro Gly Lys Gly
Leu Glu 35 40 45Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Ser
Asn Pro Ser 50 55 60Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln Phe65 70 75 80Ser Leu Thr Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Gly Tyr Asn Trp Asn Tyr
Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220132215PRTArtificial SequencegpNMB LC
132Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asp Asn
Asn 20 25 30Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Asn Asn Trp Pro Pro 85 90 95Trp Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150 155
160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu Cys 210
215133223PRTArtificial SequenceDLL3 Fd 133Gln Ile Gln Leu Val Gln
Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp
Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Ala Trp Ile
Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Lys Gly
Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Ser65 70 75
80Leu Gln Ile Ile Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95Ala Arg Ile Gly Asp Ser Ser Pro Ser Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Thr Leu Thr Val Ser Ser Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210
215 220134214PRTArtificial SequenceDLL3 LC 134Ser Ile Val Met Thr
Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30Val Val Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Tyr
Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ala Gly 50 55 60Ser
Gly Tyr Gly Thr Asp Phe Ser Phe Thr Ile Ser Thr Val Gln Ala65 70 75
80Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Thr Ser Pro Trp
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 2101356PRTArtificial SequencePeptide
linker 135Gly Gly Gly Gly Gly Ser1 51365PRTArtificial
SequenceLinker 136Ile Glu Pro Asp Ile1 51375PRTArtificial
SequenceLinker 137Leu Glu Ala Asp Thr1 51385PRTArtificial
SequenceLinker 138Ile Glu Pro Asp Gly1 51395PRTArtificial
SequenceLinker 139Ile Glu Pro Asp Val1 51405PRTArtificial
SequenceLinker 140Ile Glu Pro Asp Ser1 51415PRTArtificial
SequenceLinker 141Ile Glu Pro Asp Thr1 51424PRTArtificial
SequenceLinker 142Leu Glu Pro Asp11434PRTArtificial SequenceLinker
143Leu Glu Ala Glu11445PRTArtificial SequenceLinker 144Ile Glu Pro
Asp Pro1 5145274PRTArtificial SequenceSecond Polypeptide Chain of
B7-H3 x CD3 Bispecific DART-A Diabody 145Gln Ala Val Val Thr Gln
Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr
Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn
Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45Leu Ile Gly
Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60Ser Gly
Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75
80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly
Gly 100 105 110Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser
Gly Gly Gly 115 120 125Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly 130 135 140Phe Thr Phe Ser Ser Phe Gly Met His
Trp Val Arg Gln Ala Pro Gly145 150 155 160Lys Gly Leu Glu Trp Val
Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile 165 170 175Tyr Tyr Ala Asp
Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 180 185 190Ala Lys
Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp 195 200
205Thr Ala Val Tyr Tyr Cys Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly
210 215 220Ser Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser225 230 235 240Gly Gly Cys Gly Gly Gly Lys Val Ala Ala Leu
Lys Glu Lys Val Ala 245 250 255Ala Leu Lys Glu Lys Val Ala Ala Leu
Lys Glu Lys Val Ala Ala Leu 260 265 270Lys Glu146227PRTArtificial
SequenceThird Polypeptide Chain of B7-H3 x CD3 Bispecific DART-A
Diabody 146Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140Leu
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150
155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn Arg Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly
Lys22514718PRTArtificial SequenceLinker 147Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly1 5 10 15Gly
Ser14819PRTArtificial SequenceLinker 148Thr Gly Gly Ser Gly Gly Gly
Gly Ile Glu Pro Asp Ile Gly Gly Ser1 5 10 15Gly Gly
Ser1495PRTArtificial SequenceLinker 149Gly Gly Gly Gly Ser1
51505PRTArtificial SequenceLinker consensusVARIANT(1)..(1)X1= I, L,
Y, M, F, V, or AVARIANT(2)..(2)X2 = A, G, S, V, E, D, Q, N, or
YVARIANT(3)..(3)X3 = H, P, A, V, G, S, or TVARIANT(4)..(4)X4 = D or
EVARIANT(5)..(5)X5 = I, L, Y, M, F, V, T, S, G or A 150Xaa Xaa Xaa
Xaa Xaa1 51515PRTArtificial SequenceLinker
consensusVARIANT(1)..(1)X1 = I or LVARIANT(3)..(3)X3 = P or
AVARIANT(5)..(5)X5 = I, V, T, S, or G 151Xaa Glu Xaa Asp Xaa1
51525PRTArtificial SequenceLinker 152Leu Glu Pro Asp Gly1
51535PRTArtificial SequenceLinker 153Leu Glu Ala Asp Gly1
51545PRTArtificial SequenceLinker consensusVARIANT(1)..(1)X1 = any
amino acidVARIANT(5)..(5)X5 = A or V 154Xaa Gln Ala Arg Xaa1
51555PRTArtificial SequenceLinker consensusVARIANT(5)..(5)X5 = A or
V 155Arg Gln Ala Arg Xaa1 51565PRTArtificial SequenceLinker 156Arg
Gln Ala Arg Val1 51574PRTArtificial SequenceLinker
consensusVARIANT(1)..(1)X1 = P, V or AVARIANT(2)..(2)X2 = Q or
DVARIANT(3)..(3)X3 = A or NVARIANT(4)..(4)X4 = L, I or M 157Xaa Xaa
Xaa Xaa11584PRTArtificial SequenceLinker consensusVARIANT(2)..(2)X2
= Q or DVARIANT(3)..(3)X3 = A or NVARIANT(4)..(4)X4 = L or I 158Pro
Xaa Xaa Xaa115922PRTArtificial SequenceP2A 159Gly Ser Gly Ala Thr
Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val1 5 10 15Glu Glu Asn Pro
Gly Pro 2016019PRTArtificial SequenceP2A 160Ala Thr Asn Phe Ser Leu
Leu Lys Gln Ala Gly Asp Val Glu Glu Asn1 5 10 15Pro Gly
Pro16120PRTArtificial SequenceE2A 161Gln Cys Thr Asn Tyr Ala Leu
Leu Lys Leu Ala Gly Asp Val Glu Ser1 5 10 15Asn Pro Gly Pro
2016222PRTArtificial SequenceF2A 162Val Lys Gln Thr Leu Asn Phe Asp
Leu Leu Lys Leu Ala Gly Asp Val1 5 10 15Glu Ser Asn Pro Gly Pro
2016318PRTArtificial SequenceT2A 163Glu Gly Arg Gly Ser Leu Leu Thr
Cys Gly Asp Val Glu Glu Asn Pro1 5 10 15Gly Pro16424PRTArtificial
SequenceT2A 164Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr
Cys Gly Asp1 5 10 15Val Glu Glu Asn Pro Gly Pro Arg
2016566DNAArtificial SequenceP2A 165ggatctggag caacaaactt
ctcactactc aaacaagcag gtgacgtgga ggagaatccc 60ggaccc
6616618PRTArtificial SequenceLinker 166Gly Ser Pro Ala Gly Leu Glu
Ala Asp Gly Ser Arg Gln Ala Arg Val1 5 10 15Gly Ser167122PRTMus
musculusMISC_FEATUREanti-5T4 VHmisc_feature(97)..(97)Xaa can be any
naturally occurring amino acid 167Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Lys Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser
Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp
Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met65 70 75 80Leu Tyr
Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95Xaa
Cys Val Arg Gln Trp Asp Tyr Asp Val Arg Ala Met Asn Tyr Trp 100 105
110Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120168107PRTMus
musculusMISC_FEATUREanti-5T4 VL 168Asp Ile Val Met Thr
Gln Ser His Ile Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp
Ala Ser Thr Arg Leu Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75
80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105169504PRTArtificial SequenceFirst Polypeptide Chain of B7-H3 x
CD3 Bispecific DART-A Diabody 169Asp Ile Gln Leu Thr Gln Ser Pro
Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Gln Asn Val Asp Thr Asn 20 25 30Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile 35 40 45Tyr Ser Ala Ser Tyr
Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe 85 90 95Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Ser Gly 100 105
110Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
115 120 125Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe 130 135 140Ser Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu145 150 155 160Glu Trp Val Gly Arg Ile Arg Ser Lys
Tyr Asn Asn Tyr Ala Thr Tyr 165 170 175Tyr Ala Asp Ser Val Lys Asp
Arg Phe Thr Ile Ser Arg Asp Asp Ser 180 185 190Lys Asn Ser Leu Tyr
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr 195 200 205Ala Val Tyr
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val 210 215 220Ser
Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225 230
235 240Gly Gly Cys Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val
Ala 245 250 255Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val
Ala Ala Leu 260 265 270Glu Lys Gly Gly Gly Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala 275 280 285Pro Glu Ala Ala Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro 290 295 300Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val305 310 315 320Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 325 330 335Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 340 345
350Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
355 360 365Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala 370 375 380Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro385 390 395 400Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr 405 410 415Lys Asn Gln Val Ser Leu Trp
Cys Leu Val Lys Gly Phe Tyr Pro Ser 420 425 430Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 435 440 445Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 450 455 460Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe465 470
475 480Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys 485 490 495Ser Leu Ser Leu Ser Pro Gly Lys
50017015PRTArtificial SequenceLinker 170Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 151716PRTArtificial
SequenceLinkerREPEAT(4)..(6)repeated 0 to 10 times 171Gly Gly Ser
Gly Gly Ser1 51726PRTArtificial
SequenceLinkerREPEAT(1)..(6)repeated 1 to 4 times 172Gly Gly Gly
Gly Gly Ser1 51735PRTArtificial
SequenceLinkerREPEAT(1)..(5)Repeated 1 to 10
timesREPEAT(1)..(5)Repeated 1 to 5 times 173Gly Gly Gly Gly Ser1
51745PRTArtificial SequenceLinkerREPEAT(1)..(1)Repeated 1 to 5
timesVARIANT(2)..(2)X2= A, V, L, I, M,F, W, P, G, S, T, C, Y, N,Q,
K, R, H, D, or EREPEAT(3)..(3)Repeated 1 to 5
timesVARIANT(4)..(4)X4 = A, V, L, I, M,F, W, P, G, S, T, C, Y, N,Q,
K, R, H, D, or EREPEAT(5)..(5)Repeated 1 to 5 times 174Gly Xaa Gly
Xaa Gly1 517511PRTArtificial SequenceLinkerVARIANT(4)..(4)X4 = A,
V, L, I, M,F, W, P, G, S, T, C, Y, N,Q, K, R, H, D, or
EVARIANT(8)..(8)X8 = A, V, L, I, M,F, W, P, G, S, T, C, Y, N,Q, K,
R, H, D, or E 175Gly Gly Gly Xaa Gly Gly Gly Xaa Gly Gly Gly1 5
1017611PRTArtificial SequenceLinker 176Ala Thr Thr Thr Gly Ser Ser
Pro Gly Pro Thr1 5 1017711PRTArtificial SequenceLinker 177Gly Gly
Gly Gly Gly Cys Gly Gly Gly Gly Gly1 5 101785PRTArtificial
SequenceLinkerREPEAT(1)..(5)Repeated 2 to 20 times 178Glu Ala Ala
Ala Lys1 51796PRTArtificial SequenceLinkerREPEAT(3)..(4)Repeated 2
to 20 times 179Ala Ser Ala Pro Gly Thr1 51809PRTArtificial
SequenceLinkerREPEAT(3)..(7)Repeated 2 to 20 times 180Ala Ser Glu
Ala Ala Ala Lys Gly Thr1 51815PRTArtificial
SequenceLinkerREPEAT(1)..(5)Repeated 2 to 20 times 181Gly Gly Gly
Gly Ala1 51825PRTArtificial SequenceLinkerREPEAT(1)..(5)Repeated 2
to 20 times 182Pro Gly Gly Gly Ser1 51835PRTArtificial
SequenceLinkerREPEAT(1)..(5)Repeated 2 to 20 times 183Ala Gly Gly
Gly Ser1 518420PRTArtificial SequenceLinkerREPEAT(4)..(17)Repeated
2 to 20 times 184Gly Gly Ser Glu Gly Lys Ser Ser Gly Ser Gly Ser
Glu Ser Lys Ser1 5 10 15Thr Gly Gly Ser 201855PRTArtificial
SequenceLinkerREPEAT(1)..(5)Repeated 1 to 9 times 185Ser Ser Ser
Ser Gly1 51869PRTArtificial SequenceLinker 186Ser Ser Ser Ala Ser
Ala Ser Ser Ala1 51877PRTArtificial SequenceLinker 187Gly Ser Pro
Gly Ser Pro Gly1 5188241PRTArtificial SequenceDLL3 scFv 188Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25
30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45Gly Tyr Val Tyr Tyr Ser Gly Thr Thr Asn Tyr Asn Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95Ser Ile Ala Val Thr Gly Phe Tyr Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu 130 135 140Ser Leu Ser Pro Gly
Glu Arg Val Thr Leu Ser Cys Arg Ala Ser Gln145 150 155 160Arg Val
Asn Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln 165 170
175Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
180 185 190Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr 195 200 205Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln 210 215 220Tyr Asp Arg Ser Pro Leu Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile225 230 235 240Lys189119PRTArtificial
SequenceCD20 VH 189Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Ala Phe Ser Tyr Ser 20 25 30Trp Ile Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Phe Pro Gly Asp Gly Asp
Thr Asp Tyr Asn Gly Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val
Thr Val Ser Ser 115190115PRTArtificial SequenceCD20 VL 190Asp Ile
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25
30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 110Arg Thr Val 115191109PRTArtificial
sequenceanti-CD3 VL35 (CON) 191Gln Ala Val Val Thr Gln Glu Pro Ser
Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser
Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln
Gln Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn
Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu
Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly Ala65 70 75 80Gln Pro Glu
Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95His Trp
Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu 100
1051925PRTArtificial sequencelinker 192Gly Gly Gly Gly Gly1
519315PRTArtificial sequencelinker 193Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15194226PRTArtificial
sequenceFc-Het-1 194Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Pro Val Ala Gly1 5 10 15Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 20 25 30Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Lys His Glu 35 40 45Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His 50 55 60Asn Ala Lys Thr Lys Pro Arg Glu
Glu Glu Tyr Asn Ser Thr Tyr Arg65 70 75 80Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys 85 90 95Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 100 105 110Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 115 120 125Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 130 135
140Thr Cys Asp Val Ser Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp145 150 155 160Glu Ser Asp Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val 165 170 175Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp 180 185 190Lys Ser Arg Trp Glu Gln Gly Asp
Val Phe Ser Cys Ser Val Met His 195 200 205Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 210 215 220Gly
Lys225195226PRTArtificial sequenceFc-Het-2 195Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly1 5 10 15Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 20 25 30Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Lys His Glu 35 40 45Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 50 55 60Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg65 70 75
80Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
85 90 95Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 100 105 110Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 115 120 125Thr Leu Pro Pro Ser Arg Glu Gln Met Thr Lys
Asn Gln Val Lys Leu 130 135 140Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp145 150 155 160Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 165 170 175Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 180 185 190Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 195 200
205Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
210 215 220Gly Lys225196125PRTartificial sequenceCD3-VH32 196Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr
Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asp
Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 125197125PRTartificial sequenceCD3-VH33
197Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu
Trp Val 35 40 45Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asp Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 125198123PRTartificial
sequenceCD3-VH34 198Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Cys Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Thr Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ala Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Ser
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His
Gly Asn Phe Gly Asp Ser Tyr Val Ser Trp Phe
100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 115
120199109PRTartificial sequenceCD3-VL20 199Gln Ala Val Val Thr Gln
Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr
Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn
Trp Val Gln Gln Lys Pro Gly Lys Ser Pro Arg Gly 35 40 45Leu Ile Gly
Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser Gly
Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly Ala65 70 75
80Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105200109PRTartificial sequenceCD3-VL21 200Gln Ala Val Val Thr Gln
Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr
Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn
Trp Val Gln Gln Lys Pro Gly Lys Ser Pro Arg Gly 35 40 45Leu Ile Gly
Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser Gly
Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly Ala65 70 75
80Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95His Trp Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu 100
105201225PRTartificial sequenceKnob Fc 201Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 130 135 140Leu Trp Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200
205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220Pro225202225PRTartificial sequenceHole Fc 202Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Arg
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125Cys Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Ser Cys Ala Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220Pro225203222PRTartificial sequenceKnob Fc 203Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser1 5 10
15Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro 35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val65 70 75 80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125Pro Pro Cys Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135 140Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170
175Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
180 185 190Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 195 200 205Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 210 215 220204222PRTartificial sequenceHole Fc 204Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser1 5 10 15Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Arg Ser Arg 20 25
30Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val65 70 75 80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Cys Thr Leu 115 120 125Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140Ala Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170
175Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
180 185 190Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 195 200 205Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 210 215 220205225PRTartificial sequenceHole Fc 205Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 115 120 125Cys Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Ser Cys Ala Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170
175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 195 200 205His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220Pro225206222PRTartificial sequenceHole
Fc 206Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro
Ser1 5 10 15Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 20 25 30Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val65 70 75 80Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 115 120 125Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140Ala Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser145 150 155
160Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp
Lys Ser 180 185 190Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala 195 200 205Leu His Asn Arg Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 210 215 220207225PRTartificial sequenceKnob Fc
207Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1
5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Tyr 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro
Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Trp Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Val 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220Pro225208222PRTartificial
sequenceKnob Fc 208Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Tyr Ile Ser Arg 20 25 30Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 35 40 45Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 50 55 60Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val65 70 75 80Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125Pro
Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135
140Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser145 150 155 160Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp 165 170 175Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser 180 185 190Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Val His Glu Ala 195 200 205Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 210 215 220209225PRTartificial
sequenceHole Fc 209Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Tyr 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Val 195 200 205His Glu Ala Leu His Asn
Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220Pro225210222PRTartificial sequenceHole Fc 210Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser1 5 10 15Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val65 70 75
80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Cys Thr Leu 115 120 125Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Ser Cys 130 135 140Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175Ser Asp Gly
Ser
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His Glu Ala 195 200
205Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 210 215
22021110PRTartificial sequenceanti-CD3 VH CDR1 211Gly Phe Thr Phe
Asn Thr Tyr Ala Met Asn1 5 1021212PRTartificial sequenceanti-CD3 VH
CDR2 212Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr1 5
10213246PRTArtificial sequenceCD20 scFv 213Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30Trp Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile
Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60Lys Gly
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr
Gln Thr Pro Leu Ser 130 135 140Leu Pro Val Thr Pro Gly Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser145 150 155 160Lys Ser Leu Leu His Ser
Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu 165 170 175Gln Lys Pro Gly
Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn 180 185 190Leu Val
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200
205Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val
210 215 220Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly
Gly Gly225 230 235 240Thr Lys Val Glu Ile Lys
245214118PRTArtificial sequenceB7H3 sdAb B7h3 hz1A5v51 214Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Asn 20 25
30Val Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile Tyr Ser Ser Gly Thr Gly Thr Phe Tyr Ala Glu Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Thr Ser Gly Pro Val Arg Gly Trp Gly Pro
Arg Ser Gln Gly Thr 100 105 110Leu Val Thr Val Lys Pro
115215117PRTartificial sequencesdAb B7H3 hz58E05v27 215Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ser Tyr 20 25 30His
Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Pro Val 35 40
45Ala Thr Ser His His Gly Gly Thr Thr Asn Tyr Ala Gly Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
Leu65 70 75 80Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Lys 85 90 95Ala Asp His Gly Tyr Gln Gly Arg Gly Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Lys Pro
115216117PRTartificial sequencesdAb B7H3 hz58E05v55 216Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30His
Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45Ala Thr Ser His His Gly Gly Thr Thr Asn Tyr Ala Gly Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
Leu65 70 75 80Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Lys 85 90 95Ala Asp His Gly Tyr Gln Gly Arg Gly Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Lys Pro
115217117PRTartificial sequencesdAb B7H3 hz58E05v48 217Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30His
Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Pro Val 35 40
45Ala Thr Ser His His Gly Gly Thr Thr Asn Tyr Ala Gly Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
Leu65 70 75 80Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Lys 85 90 95Ala Asp His Gly Tyr Gln Gly Arg Gly Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Lys Pro
115218127PRTartificial sequencesdAb B7H3 hz57B04v24 218Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Pro Ser Glu Arg Thr Phe Ser Thr Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Val Val Asn Trp Gly Gly Gly Ser Lys Tyr Tyr Ala Glu Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Ala Gly Gly Ala Tyr Ser Gly Pro Tyr Tyr Asp
Thr Arg Gln Tyr 100 105 110Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Lys Pro Gly Gly 115 120 125219120PRTartificial sequencesdAb
DLL3 hz10D9v7 219Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
Ile Phe Ser Ile Asn 20 25 30Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly
Lys Gln Arg Glu Leu Val 35 40 45Ala Gly Phe Thr Gly Asp Thr Asn Thr
Ile Tyr Ala Glu Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Ser Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Asp Val Gln Leu
Phe Ser Arg Asp Tyr Glu Phe Tyr Trp Gly Gln 100 105 110Gly Thr Leu
Val Thr Val Lys Pro 115 120220115PRTartificial sequencesdAb DLL3
hz8E7v16 220Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Gly Pro Ser Glu Ile Ile Thr
Ser Asp Lys 20 25 30Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gln
Arg Asn Leu Val 35 40 45Ala Gly Ile Ser Asn Val Gly Ser Thr Asn Tyr
Ala Gln Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Ser Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95Ala Arg Asp Phe Glu Asn Glu
Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Lys Pro
11522110PRTartificial sequence41BB CDR1 221Gly Phe Ser Phe Ser Ile
Asn Ala Met Gly1 5 102229PRTartificial sequence41BB CDR2 222Ala Ile
Glu Ser Gly Arg Asn Thr Val1 522313PRTartificial sequence41BB CDR3
223Leu Lys Gly Asn Arg Val Val Ser Pro Ser Val Ala Tyr1 5
1022414PRTartificial sequenceCD3-VH7, VH33 CDR3 224His Gly Asn Phe
Gly Asp Ser Tyr Val Ser Trp Phe Ala Tyr1 5 102259PRTartificial
sequenceCD3-VL2, VL21 CDR3 225Ala Leu Trp Tyr Ser Asn His Trp Val1
52269PRTartificial sequenceCD3-VL8 CDR3 226Val Leu Trp Tyr Ser Asn
Arg Trp Val1 522710PRTartificial sequenceCD3 VH33 CDR1 227Gly Phe
Thr Phe Ser Thr Tyr Ala Met Asn1 5 1022812PRTartificial sequenceCD3
VH33 CDR2 228Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr1 5
1022914PRTartificial sequenceCD3 VL21 CDR1 229Gly Ser Ser Thr Gly
Ala Val Thr Thr Ser Asn Tyr Ala Asn1 5 102307PRTartificial
sequenceCD3 VL21 CDR2 230Gly Thr Asn Lys Arg Ala Pro1 5
* * * * *
References