U.S. patent application number 14/774649 was filed with the patent office on 2016-01-21 for bispecific antibodies and uses thereof.
The applicant listed for this patent is THE CALIFORNIA INSTITUTE FOR BIOMEDICAL RESEARCH, THE SCRIPPS RESEARCH INSTITUTE. Invention is credited to Jun Y. AXUP, Yu CAO, Chanhyuk KIM, Stephanie PINKERTON, Peter G. SCHULTZ, Travis YOUNG, Quan ZHOU.
Application Number | 20160017058 14/774649 |
Document ID | / |
Family ID | 51581385 |
Filed Date | 2016-01-21 |
United States Patent
Application |
20160017058 |
Kind Code |
A1 |
KIM; Chanhyuk ; et
al. |
January 21, 2016 |
BISPECIFIC ANTIBODIES AND USES THEREOF
Abstract
Methods, compositions and uses are provided for bispecific
antibodies comprising one or more unnatural amino acids. The
bispecific antibodies may bind to two or more different receptors,
co-receptors, antigens, or cell markers on one or more cells. The
bispecific antibodies may be used to treat a disease or condition
(e.g., cancer, autoimmune disease, pathogenic infection,
inflammatory disease). The bispecific antibodies may be used to
modulate (e.g., stimulate or suppress) an immune response.
Inventors: |
KIM; Chanhyuk; (San Diego,
CA) ; AXUP; Jun Y.; (San Diego, CA) ; SCHULTZ;
Peter G.; (La Jolla, CA) ; YOUNG; Travis; (La
Jolla, CA) ; PINKERTON; Stephanie; (San Diego,
CA) ; ZHOU; Quan; (Somerset, NJ) ; CAO;
Yu; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE CALIFORNIA INSTITUTE FOR BIOMEDICAL RESEARCH
THE SCRIPPS RESEARCH INSTITUTE |
La Jolla
La Jolla |
CA
CA |
US
US |
|
|
Family ID: |
51581385 |
Appl. No.: |
14/774649 |
Filed: |
March 14, 2014 |
PCT Filed: |
March 14, 2014 |
PCT NO: |
PCT/US2014/028612 |
371 Date: |
September 10, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61783426 |
Mar 14, 2013 |
|
|
|
61839330 |
Jun 25, 2013 |
|
|
|
Current U.S.
Class: |
424/136.1 ;
530/387.3 |
Current CPC
Class: |
C07K 16/468 20130101;
C07D 249/04 20130101; A61K 47/542 20170801; A61P 35/02 20180101;
C07K 16/2887 20130101; C07K 16/32 20130101; A61K 39/39558 20130101;
C07K 16/3069 20130101; C07K 16/2863 20130101; A61P 17/00 20180101;
C07C 275/24 20130101; C07K 2317/73 20130101; C07K 2317/31 20130101;
A61P 35/00 20180101; A61K 39/3955 20130101; C07K 16/2851 20130101;
A61K 2039/505 20130101; C07K 16/2803 20130101; C07K 2317/92
20130101; Y02A 50/30 20180101; C07K 16/2896 20130101; C07K 2317/55
20130101; C07K 2317/56 20130101; C07K 2317/35 20130101; C07K
2317/52 20130101; C07K 2317/33 20130101; C07K 2317/94 20130101;
C07C 275/16 20130101; C07K 16/2809 20130101; C07K 16/2806
20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/28 20060101 C07K016/28 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with the support of the United
States government under Contract number GM062159 by National
Institutes of Health (NIH). The government has certain rights in
the invention.
Claims
1. A bispecific antibody comprising: a. an anti-CD3 antibody or
anti-CD3 antibody fragment; b. a second antibody or antibody
fragment, wherein the anti-CD3 antibody or anti-CD3 antibody
fragment is site-specifically connected to the second antibody or
antibody fragment.
2. The bispecific antibody of claim 1, wherein the anti-CD3
antibody or anti-CD3 antibody fragment is site-specifically
connected to the second antibody or antibody fragment by one or
more linkers.
3. The bispecific antibody of claim 1, wherein the second antibody
or antibody fragment comprises an anti-EGFRvIII antibody.
4. The bispecific antibody of claim 1, wherein the second antibody
or antibody fragment comprises an anti-Her2 antibody.
5. The bispecific antibody of claim 1, wherein the second antibody
or antibody fragment comprises an anti-CS1 antibody.
6. The bispecific antibody of claim 1, wherein the second antibody
or antibody fragment comprises an anti-CLL-1 antibody.
7. The bispecific antibody of claim 1, wherein the second antibody
or antibody fragment comprises an anti-CD33 antibody.
8. The bispecific antibody of claim 1, wherein the second antibody
or antibody fragment comprises an anti-CD20 antibody.
9. The bispecific antibody of claim 1, wherein the second antibody
or antibody fragment is selected from an anti-ROR1 antibody, an
anti-CD44v6 antibody, an anti-PVRL4 antibody, an anti-IL13R.alpha.2
antibody and a bscWue1 antibody.
10. The bispecific antibody of claim 1, wherein the anti-CD3
antibody or anti-CD3 antibody fragment comprises one or more
unnatural amino acids.
11. The bispecific antibody of claim 1, wherein the second antibody
or antibody fragment comprises one or more unnatural amino
acids.
12. The bispecific antibody of claim 1, wherein the anti-CD3
antibody or anti-CD3 antibody fragment and the second antibody or
antibody fragment comprise one or more unnatural amino acids.
13. The bispecific antibody of claim 2, wherein the antibody is of
Formula I: X-L1'-Y or Formula IA: Y-L1'-X, wherein: a. X comprises
the anti-CD3 antibody or anti-CD3 antibody fragment; b. L1'
comprises the one or more linkers; and c. Y comprises the second
antibody or antibody fragment.
14. The bispecific antibody of claim 13, wherein X is selected from
a human, humanized, human engineered or fully human antibody.
15. The bispecific antibody of claim 1, wherein X comprises a
chimeric antibody or portion thereof.
16. The bispecific antibody of claim 1, wherein X comprises a
cross-species reactive antibody or a portion thereof.
17. The bispecific antibody of claim 16, wherein the anti-CD3
antibody is cross-species reactive with human and cynomolgus
monkey.
18. The bispecific antibody of claim 13, wherein X and/or Y
comprises one or more Fv, Fc, Fab, (Fab')2, single chain Fv (scFv),
diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies,
CDR1, CDR2, CDR3, combinations of CDR's, variable regions,
framework regions, constant regions, heavy chains, light chains,
and variable regions, alternative scaffold non-antibody molecules,
or a combination thereof.
19. The bispecific antibody of claim 13, wherein X and/or Y
comprises a Fab fragment.
20. The bispecific antibody of claim 19, wherein the anti-CD3
antibody fragment is UCHT1.
21. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of a Fab fragment.
22. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of an antibody or antibody fragment that binds to
an antigen on a hematopoietic cell.
23. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of an antibody or antibody fragment that binds to
an antigen on a myeloid cell.
24. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of an antibody or antibody fragment that binds to
an antigen on a lymphocyte.
25. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of an antibody or antibody fragment that binds to
an antigen on a B-cell or B-cell progenitor.
26. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of an antibody or antibody fragment that binds to
an antigen on a cancerous cell.
27. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of an antibody selected from the group comprising
an anti-CD20 antibody, an anti-EGFRvIII antibody, an anti-CS1
antibody, an anti-CLL-1 antibody and an anti-CD33 antibody.
28. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of a Fab fragment of an antibody selected from the
group comprising an anti-CD20 antibody, an anti-EGFRvIII antibody,
an anti-CS1 antibody, an anti-CLL-1 antibody and an anti-CD33
antibody.
29. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of an antibody selected from the group comprising
an anti-ROR1 antibody, an anti-CD44v6 antibody, an anti-PVRL4
antibody, an anti-IL13R.alpha.2 antibody and an anti-bscWue1
antibody.
30. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of Fab fragment of an antibody selected from the
group comprising an anti-ROR1 antibody, an anti-CD44v6 antibody, an
anti-PVRL4 antibody, an anti-IL13R.alpha.2 antibody and a bscWue1
antibody.
31. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of an anti-Her2 antibody.
32. The bispecific antibody of claim 13, wherein Y comprises at
least a portion of a Fab fragment of an anti-Her2 antibody.
33. The bispecific antibody of claim 13, wherein Y is selected form
SEQ ID NOs: 3-16.
34. The bispecific antibody of claim 13, wherein the bispecific
antibody further comprises a second linker.
35. The bispecific antibody of claim 34, wherein the bispecific
antibody is of Formula II: X-L1-L2-Y or Formula IIA: Y-L2-L1-X,
wherein: a. a L1'' is coupled to X to produce a first intermediate
of Formula III: X-L1' or Formula IIIA: L1'-X, wherein X comprises
an anti-CD3 antibody or anti-CD3 antibody fragment; and L1''
comprises a first linker before being coupled to X, b. L2'' is
coupled to Y to produce a second intermediate of Formula IV: Y-L2'
or Formula IVA: L2'-Y; wherein Y comprises at least a portion of a
second antibody or antibody fragment and L2'' comprises a second
linker before being coupled to Y; and c. the first intermediate is
coupled to the second intermediate.
36. The bispecific antibody of claim 35, wherein L1'' and/or L2''
comprise one or more chemical groups selected from an alkoxy-amine,
hydrazine, aryl azide, alkyl azide, alkyne, alkene, tetrazine,
dichlorotriazine, tresylate, succinimidyl carbonate, benzotriazole
carbonate, nitrophenyl carbonate, trichlorophenyl carbonate,
carbonylimidazole, succinimidyl succinate, maleimide, vinylsulfone,
haloacetamide, cyclooctyne, trans-cyclooctene, cyclopropene,
norborene and disulfide.
37. The bispecific antibody of claim 35, wherein one terminus of
L1'' and/or one terminus of L2'' comprises an alkoxy-amine.
38. The bispecific antibody of claim 35, wherein one terminus of
L1'' and/or one terminus of L2'' comprises an azide or cyclooctyne
group.
39. The bispecific antibody of claim 35, wherein X is coupled to L1
by a covalent bond, ionic bond, or non-covalent bond.
40. The bispecific antibody of claim 35, wherein Y is coupled to L2
by a covalent bond, ionic bond, or non-covalent bond.
41. The bispecific antibody of claim 35, wherein X is coupled to L1
by an oxime.
42. The bispecific antibody of claim 35, wherein Y is coupled to L2
by an oxime.
43. The bispecific antibody of claim 35, wherein X-L1' and L2-Y'
are linked through a reaction selected from a
Huisgen-cycloaddition, a Diels-Halder reaction, a hetero
Diels-Alder reaction and an enzyme-mediated reaction.
44. The bispecific antibody of claim 43, wherein the
enzyme-mediated reaction comprises a transglutaminase reaction.
45. The bispecific antibody of claim 35, wherein X-L1' and Y-L2'
are linked through a copper-free [3+2] Huisgen-cycloaddition.
46. The bispecific antibody of claim 35, wherein X-L1' and L2'-Y
are linked through a disulfide or a maleimide.
47. The bispecific antibody of claim 35, wherein the distance
between X and Y is less than about 50 angstrom (.ANG.), about 45
angstrom (.ANG.), about 40 angstrom (.ANG.), about 35 angstrom
(.ANG.), about 30 angstrom (.ANG.), about 25 angstrom (.ANG.),
about 20 angstrom (.ANG.), about 15 angstrom (.ANG.), about 10
angstrom (.ANG.) or about 5 angstrom (.ANG.).
48. The bispecific antibody of claim 2, wherein the one or more
linkers comprises one or more ethylene glycol subunits.
49. The bispecific antibody of claim 13, wherein X and/or Y
comprise one or more unnatural amino acids.
50. The bispecific antibody of claim 49, wherein one or more
unnatural amino acids are site-specifically incorporated into X
and/or Y.
51. The bispecific antibody of claim 49, wherein the one or more
unnatural amino acids of X and/or the one or more unnatural amino
acids of Y are genetically encoded.
52. The bispecific antibody of claim 51, wherein the one or more
unnatural amino acids of X and/or the one or more unnatural amino
acids of Y are genetically encoded by a codon selected from a
nonsense codon, a stop codon, an umber codon, an ochre codon, an
opal codon, a four-base codon and an amber codon.
53. The bispecific antibody of claim 49, wherein the one or more
unnatural amino acids of X and/or the one or more unnatural amino
acids of Y comprise a p-acetylphenylalanine (pAcF).
54. The bispecific antibody of claim 49, wherein the one or more
unnatural amino acids of X and/or the one or more unnatural amino
acids of Y comprise a selenocysteine.
55. The bispecific antibody of claim 49, wherein the site-specific
connection occurs via the one or more unnatural amino acids of X
and/or Y.
56. A method of producing a bispecific antibody, the method
comprising connecting a plurality of anti-CD3 antibodies or
anti-CD3 antibody fragments to a plurality of second antibodies or
antibody fragments to produce a plurality of anti-CD3 bispecific
antibodies, wherein at least 60% of the anti-CD3 bispecific
antibodies are identical.
57. A method of producing a bispecific antibody of Formula II:
X-L1'-L2'-Y or Formula HA: Y-L2'-L1'-X, comprising: d. coupling
L1'' to X to produce a first intermediate of Formula III: X-L1' or
Formula IIIA: L1'-X, wherein X comprises an anti-CD3 antibody or
anti-CD3 antibody fragment; and L1'' comprises a first linker
before being coupled to X, e. coupling L2'' to Y to produce a
second intermediate of Formula IV: Y-L2' or Formula IVA: L2'-Y;
wherein Y comprises at least a portion of a second antibody or
antibody fragment and L2'' comprises a second linker before being
coupled to Y; and f. linking the first intermediate to the second
intermediate, thereby producing the antibody of Formula II or
IIA.
58. The method of claim 57, wherein coupling L1'' to X comprises
site-specific coupling of L1'' to X and/or coupling L2'' to Y
comprises site-specific coupling of L2'' to Y.
59. The method of claim 57, further comprising incorporating one or
more unnatural amino acids into X and/or Y.
60. The method of claim 59, wherein coupling L1'' to X occurs at
the one or more unnatural amino acids in X and/or coupling L2'' to
Y occurs at the one or more unnatural amino acids in Y.
61. The method of claim 59, further comprising modifying a nucleic
acid encoding X and/or Y to produce one or more amber codons in X
and/or Y.
62. The method of claim 57, wherein linking the first intermediate
to the second intermediate comprises a Huisgen-cycloaddition, a
Diels-Halder reaction, a hetero Diels-Alder reaction or an
enzyme-mediated reaction
63. The method of claim 57, wherein linking the first intermediate
to the second intermediate comprises a copper-free [3+2]
Huisgen-cycloaddition reaction.
64. The method of claim 57, wherein linking the first intermediate
to the second intermediate produces an oxime, a tetrazole, a Diels
Alder adduct, a hetero Diels Alder adduct, an aromatic substitution
reaction product, a nucleophilic substitution reaction product, an
ester, an amide, a carbamate, an ether, a thioether, a Michael
reaction product, cycloaddition product, a metathesis reaction
product, a metal-mediated cross-coupling reaction product, a
radical polymerization product, an oxidative coupling product, an
acyl-transfer reaction product, or a photo click reaction
product.
65. The method of claim 57, wherein linking the first intermediate
to the second intermediate produces a disulfide bridge or a
maleimide bridge.
66. The method of claim 57, wherein the distance between X and Y is
less than or equal to 50, 45, 40, 35, 30, 25, or 20 angstroms
(.ANG.).
67. The method of claim 57, wherein the distance between X and Y is
greater than or equal to 5 angstroms (.ANG.).
68. A pharmaceutical composition comprising the bispecific antibody
of any of claims 1-54.
69. The pharmaceutical composition of claim 68, further comprising
a pharmaceutically acceptable diluent, a pharmaceutically
acceptable excipient or a pharmaceutically acceptable carrier.
70. A method for treating a disease or condition in a subject in
need thereof, comprising administering the bispecific antibody of
any of claims 1-55 or the pharmaceutical composition of any of
claims 68 and 69.
71. The method of claim 70, wherein the disease or condition is a
cancer.
72. The method of claim 71, wherein the cancer is a breast
cancer.
73. The method of claim 71, wherein the cancer is a glioma or
glioblastoma.
74. The method of claim 71, wherein the cancer is a multiple
myeloma.
75. The method of claim 71, wherein the cancer is an acute myeloid
leukemia (AML).
76. The method of claim 71, wherein the cancer is selected from an
acute lymphoblastic leukemia (ALL), a B-cell chronic lymphocytic
leukemia (B-CLL) and a mantle cell lymphoma (MCL).
77. The method of claim 70, wherein the disease or condition is a
pathogenic infection.
78. The method of claim 70, wherein the disease or condition is an
inflammatory disease, an autoimmune disease or a metabolic
disease.
79. The method of claim 70, wherein the bispecific antibody or
pharmaceutical composition is administered by parenteral
administration.
80. The method of claim 79, wherein parenteral administration
comprises intravenous administration, subcutaneous administration,
intraperitoneal administration, intramuscular administration,
intravascular administration, intrathecal administration,
intravitreal administration, or infusion.
81. The method of claim 70, wherein the bispecific antibody is
administered by a microneedle device.
82. The method of claim 70, wherein the bispecific antibody is
administered by topical, oral, or nasal administration.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. provisional
application Ser. No. 61/783,426, filed Mar. 14, 2013 and U.S.
provisional application Ser. No. 61/839,330, filed Jun. 25, 2013;
which are incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] Described herein are immunoglobulin constructs comprising
one or more unnatural amino acids, methods of making such
constructs, pharmaceutical compositions and medicaments comprising
such constructs, and methods of using such constructs and
compositions to prevent, inhibit, and/or treat a disease or
condition in a subject.
BACKGROUND OF THE INVENTION
[0004] Antibodies are natural proteins that the vertebrate immune
system forms in response to foreign substances (antigens),
primarily for defense against infection. For over a century,
antibodies have been induced in animals under artificial conditions
and harvested for use in therapy or diagnosis of disease
conditions, or for biological research. Each individual antibody
producing cell produces a single type of antibody with a chemically
defined composition, however, antibodies obtained directly from
animal serum in response to antigen inoculation actually comprise
an ensemble of non-identical molecules (e.g., polyclonal
antibodies) made from an ensemble of individual antibody producing
cells.
[0005] Some immunoglobulin constructs, such as bispecific
antibodies, may bind to two or more different antigens. A number of
recombinant strategies have been developed to synthesize bispecific
antibodies, which include single chain variable fragment
(scFv)-derived formats such as diabodies, tandem diabodies, BiTes
(bispecific T-cell engager), and DARTs (Dual Affinity
Re-Targeting), as well as immunoglobulin G (IgG)-based formats such
as Triomab, DVD-Ig (Dual Variable Domain antibodies), and
two-in-one antibodies. In addition, a number of chemical approaches
have been developed which largely exploit the reactivity of lysine
or cysteine residues within the antibody. Another chemical strategy
has been reported in which heterodimeric peptides with a branched
azetidinone linker were fused to the antibody in a site-specific
manner. These current approaches may result in heterogeneous
products and/or may require the development of antigen-specific
ligands, rather than utilizing the diverse pool of existing
selective, high affinity monoclonal antibodies.
[0006] Disclosed herein are immunoglobulin constructs comprising
one or more unnatural amino acids, methods of producing such
constructs, and uses thereof.
SUMMARY OF THE INVENTION
[0007] Disclosed herein are bispecific antibodies comprising: an
anti-CD3 antibody or anti-CD3 antibody fragment; a second antibody
or antibody fragment, wherein the anti-CD3 antibody or anti-CD3
antibody fragment is site-specifically connected to the second
antibody or antibody fragment. The anti-CD3 antibody or anti-CD3
antibody fragment may be site-specifically connected to the second
antibody or antibody fragment by one or more linkers. The second
antibody or antibody fragment may comprise an anti-EGFRvIII
antibody. The second antibody or antibody fragment may comprise an
anti-Her2 antibody. The second antibody or antibody fragment may
comprise an anti-CS1 antibody. The second antibody or antibody
fragment may comprise an anti-CLL-1 antibody. The second antibody
or antibody fragment may comprise an anti-CD33 antibody. The second
antibody or antibody fragment may comprise an anti-CD20 antibody.
The second antibody or antibody fragment may comprise an anti-ROR1
antibody. The second antibody or antibody fragment may comprise an
anti-CD44v6 antibody. The second antibody or antibody fragment may
comprise an anti-PVRL4 antibody. The second antibody or antibody
fragment may comprise an anti-IL13R.alpha.2 antibody. The second
antibody or antibody fragment may comprise a bscWue1 antibody. The
anti-CD3 antibody or anti-CD3 antibody fragment may comprise one or
more unnatural amino acids. The second antibody or antibody
fragment comprises one or more unnatural amino acids. The anti-CD3
antibody or anti-CD3 antibody fragment and the second antibody or
antibody fragment may comprise one or more unnatural amino
acids.
[0008] The bispecific antibody may be of a Formula I: X-L1'-Y or a
Formula IA: Y-L1'-X, wherein: X comprises the anti-CD3 antibody or
anti-CD3 antibody fragment; L1' comprises the one or more linkers;
and Y comprises the second antibody or antibody fragment. X may
comprise a human antibody or human antibody fragment. X may
comprise a humanized antibody or humanized antibody fragment. X may
comprise a human engineered antibody or human engineered antibody
fragment. X may comprise a fully human antibody or fully human
antibody fragment. X may comprise a chimeric antibody or portion
thereof. X may comprise a cross-species reactive antibody or a
portion thereof. The anti-CD3 antibody may be cross-species
reactive with human and cynomolgus monkey. X and/or Y may comprise
one or more Fv. X and/or Y may comprise one or more Fc. X and/or Y
may comprise one or more Fab. X and/or Y may comprise one or more
(Fab')2. X and/or Y may comprise one or more single chain Fv
(scFv). X and/or Y may comprise one or more diabodies. X and/or Y
may comprise one or more triabodies. X and/or Y may comprise one or
more tetrabodies. X and/or Y may comprise one or more bifunctional
hybrid antibodies. X and/or Y may comprise one or more CDR1. X
and/or Y may comprise one or more CDR2. X and/or Y may comprise one
or more CDR3. X and/or Y may comprise one or more combinations of
CDR's. X and/or Y may comprise one or more variable regions. X
and/or Y may comprise one or more framework regions. X and/or Y may
comprise one or more constant regions. X and/or Y may comprise one
or more heavy chains. X and/or Y may comprise one or more light
chains. X and/or Y may comprise one or more and variable regions. X
and/or Y may comprise one or more alternative scaffold non-antibody
molecules. X and/or Y may comprise a combination of Fv, Fc, Fab,
(Fab')2, single chain Fv (scFv), diabodies, triabodies,
tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3,
combinations of CDR's, variable regions, framework regions,
constant regions, heavy chains, light chains, and variable regions,
alternative scaffold non-antibody molecules. X and/or Y may
comprise a Fab fragment. The anti-CD3 antibody fragment may be
UCHT1. Y may comprise at least a portion of a Fab fragment. Y may
comprise at least a portion of an antibody or antibody fragment
that binds to an antigen on a hematopoietic cell. Y may comprise at
least a portion of an antibody or antibody fragment that binds to
an antigen on a myeloid cell. Y may comprise at least a portion of
an antibody or antibody fragment that binds to an antigen on a
lymphocyte. Y may comprise at least a portion of an antibody or
antibody fragment that binds to an antigen on a B-cell or B-cell
progenitor. Y may comprise at least a portion of an antibody or
antibody fragment that binds to an antigen on a cancerous cell. Y
may comprise at least a portion of an antibody selected from the
group comprising an anti-CD20 antibody, an anti-EGFRvIII antibody,
an anti-CS1 antibody, an anti-CLL-1 antibody and an anti-CD33
antibody. Y may comprise at least a portion of a Fab fragment of an
antibody selected from the group comprising an anti-CD20 antibody,
an anti-EGFRvIII antibody, an anti-CS1 antibody, an anti-CLL-1
antibody and an anti-CD33 antibody. Y may comprise at least a
portion of an antibody selected from the group comprising an
anti-ROR1 antibody, an anti-CD44v6 antibody, an anti-PVRL4
antibody, an anti-IL13R.alpha.2 antibody and an anti-bscWue1
antibody. Y may comprise at least a portion of Fab fragment of an
antibody selected from the group comprising an anti-ROR1 antibody,
an anti-CD44v6 antibody, an anti-PVRL4 antibody, an
anti-IL13R.alpha.2 antibody and a bscWue1 antibody. Y may comprise
at least a portion of an anti-Her2 antibody. Y may comprise at
least a portion of a Fab fragment of an anti-Her2 antibody. Y may
be selected form SEQ ID NOs: 3-16.
[0009] The bispecific antibody may further comprise a second
linker. The bispecific antibody may be of Formula II: X-L1-L2-Y or
Formula IIA: Y-L2-L1-X, wherein: a L1'' is coupled to X to produce
a first intermediate of Formula III: X-L1' or Formula IIIA: L1'-X,
wherein X comprises an anti-CD3 antibody or anti-CD3 antibody
fragment; and L1'' comprises a first linker before being coupled to
X; L2'' is coupled to Y to produce a second intermediate of Formula
IV: Y-L2' or Formula IVA: L2'-Y; wherein Y comprises at least a
portion of a second antibody or antibody fragment and L2''
comprises a second linker before being coupled to Y; and the first
intermediate is coupled to the second intermediate. L1'' may
comprise one or more chemical groups selected from an alkoxy-amine,
hydrazine, aryl azide, alkyl azide, alkyne, alkene, tetrazine,
dichlorotriazine, tresylate, succinimidyl carbonate, benzotriazole
carbonate, nitrophenyl carbonate, trichlorophenyl carbonate,
carbonylimidazole, succinimidyl succinate, maleimide, vinylsulfone,
haloacetamide, cyclooctyne, trans-cyclooctene, cyclopropene,
norborene and disulfide. L2'' may comprise one or more chemical
groups selected from an alkoxy-amine, hydrazine, aryl azide, alkyl
azide, alkyne, alkene, tetrazine, dichlorotriazine, tresylate,
succinimidyl carbonate, benzotriazole carbonate, nitrophenyl
carbonate, trichlorophenyl carbonate, carbonylimidazole,
succinimidyl succinate, maleimide, vinylsulfone, haloacetamide,
cyclooctyne, trans-cyclooctene, cyclopropene, norborene and
disulfide. One terminus of L1'' may comprise an alkoxy-amine. One
terminus of L2'' may comprise an alkoxy-amine. One terminus of L1''
may comprise an azide group. One terminus of L1'' may comprise an
cyclooctyne group. One terminus of L2'' may comprise an azide
group. One terminus of L2'' may comprise an cyclooctyne group. X
may be coupled to L1 by a covalent bond, ionic bond, or
non-covalent bond. Y may be coupled to L2 by a covalent bond, ionic
bond, or non-covalent bond. X may coupled to L1 by an oxime. Y may
be coupled to L2 by an oxime. X-L1' and L2'-Y may be linked through
a reaction selected from a Huisgen-cycloaddition. L2'-Y may be
linked through a reaction selected from a Diels-Halder reaction.
L2'-Y may be linked through a reaction selected from a hetero
Diels-Alder reaction and an enzyme-mediated reaction. L1'-X and
Y-L2' may be linked through a reaction selected from a
Huisgen-cycloaddition. L1'-X and Y-L2' may be linked through a
reaction selected from a Diels-Halder reaction. L1'-X and Y-L2' may
be linked through a reaction selected from a hetero Diels-Alder
reaction. L1'-X and Y-L2' may be linked through a reaction selected
from an enzyme-mediated reaction. The enzyme-mediated reaction may
comprise a transglutaminase reaction. X-L1' and L2'-Y may be linked
through a copper-free [3+2] Huisgen-cycloaddition. Y-L2' and L1'-X
may be linked through a copper-free [3+2] Huisgen-cycloaddition.
X-L1' and L2'-Y may be linked through a disulfide or a maleimide.
Y-L2' and L1'-X may be linked through a disulfide or a maleimide.
The distance between X and Y may be less than about 50 angstrom
(.ANG.), about 45 angstrom (.ANG.), about 40 angstrom (.ANG.),
about 35 angstrom (.ANG.), about 30 angstrom (.ANG.), about 25
angstrom (.ANG.), about 20 angstrom (.ANG.), about 15 angstrom
(.ANG.), about 10 angstrom (.ANG.) or about 5 angstrom (.ANG.). The
one or more linkers may comprise one or more ethylene glycol
subunits.
[0010] The bispecific antibodies disclosed herein may comprise one
or more unnatural amino acids. X may comprise one or more unnatural
amino acids. Y may comprise one or more unnatural amino acids. The
one or more unnatural amino acids may be site-specifically
incorporated into X. The one or more unnatural amino acids may be
site-specifically incorporated into Y. The one or more unnatural
amino acids of X may be genetically encoded. The one or more
unnatural amino acids of Y may be genetically encoded. The one or
more unnatural amino acids of X may be genetically encoded by a
codon selected from a nonsense codon, a stop codon, an umber codon,
an ochre codon, an opal codon, a four-base codon and an amber
codon. The one or more unnatural amino acids of Y may be
genetically encoded by a codon selected from a nonsense codon, a
stop codon, an umber codon, an ochre codon, an opal codon, a
four-base codon and an amber codon. The one or more unnatural amino
acids of X may comprise a p-acetylphenylalanine (pAcF). The one or
more unnatural amino acids of Y may comprise a
p-acetylphenylalanine (pAcF). The one or more unnatural amino acids
of X may comprise selenocysteine. The one or more unnatural amino
acids of Y may comprise a selenocysteine. The one or more unnatural
amino cids may be p-fluorophenylalanine (pFPhe). The one or more
unnatural amino acids may be selected from the group comprising
p-azidophenylalanine (pAzF), p-benzoylphenylalanine (pBpF),
p-propargyloxyphenylalanine (pPrF), p-iodophenylalanine (pIF),
p-cyanophenylalanine (pCNF), p-carboxylmethylphenylalanine (pCmF),
3-(2-naphthyl)alanine (NapA), p-boronophenylalanine (pBoF),
o-nitrophenylalanine (oNiF), (8-hydroxyquinolin-3-yl)alanine (HQA),
selenocysteine, and (2,2'-bipyridin-5-yl)alanine (BipyA). The one
or more unnatural amino acids may be .beta.-amino acids (.beta.3
and .beta.2), homo-amino acids, proline and pyruvic acid
derivatives, 3-substituted alanine derivatives, glycine
derivatives, ring-substituted phenylalanine and tyrosine
derivatives, linear core amino acids, diamino acids, D-amino acids,
N-methyl amino acids, or a combination thereof. The site-specific
connection may occur via the one or more unnatural amino acids of
X. The site-specific connection may occur via the one or more
unnatural amino acids of Y.
[0011] The purity of the bispecific antibody may be equal to or
greater than 50%. The purity of the bispecific antibody may be
equal to or greater than 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or
98%. The homogeneity of the bispecific antibody may be equal to or
greater than 50%. The homogeneity of the bispecific antibody may be
equal to or greater than 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or
98%.
[0012] Further disclosed herein are methods of producing bispecific
antibodies, the method comprising connecting a plurality of
anti-CD3 antibodies or anti-CD3 antibody fragments to a plurality
of second antibodies or antibody fragments to produce a plurality
of anti-CD3 bispecific antibodies, wherein at least 60% of the
anti-CD3 bispecific antibodies are identical.
[0013] Disclosed herein are methods of producing a bispecific
antibodies of Formula II: X-L1'-L2'-Y or Formula IIA: Y-L2'-L1'-X,
comprising: coupling L1'' to X to produce a first intermediate of
Formula III: X-L1' or Formula IIIA: L1'-X, wherein X comprises an
anti-CD3 antibody or anti-CD3 antibody fragment; and L1'' comprises
a first linker before being coupled to X, coupling L2'' to Y to
produce a second intermediate of Formula IV: Y-L2' or Formula IVA:
L2'-Y; wherein Y comprises at least a portion of a second antibody
or antibody fragment and L2'' comprises a second linker before
being coupled to Y; and linking the first intermediate to the
second intermediate, thereby producing the antibody of Formula II
or HA. Coupling L1'' to X may comprise site-specific coupling of
L1'' to X. Coupling L2'' to Y may comprise site-specific coupling
of L2'' to Y. The method may further comprise incorporating one or
more unnatural amino acids into X. The method may further comprise
incorporating one or more unnatural amino acids into Y.
Incorporating one or more unnatural amino acids into X and/or Y may
comprise modifying one or more amino acid residues in X and/or Y to
produce one or more amber codons in X and/or Y. The one or more
unnatural amino acids may be incorporated into X and/or Y in
response to an amber codon. The one or more unnatural amino acids
may be site-specifically incorporated into X and/or Y.
Incorporating one or more unnatural amino acids may comprise the
use of one or more tRNA synthetases. The tRNA synthetase may be an
aminoacyl tRNA synthetase. The tRNA synthetase may be a mutant tRNA
synthesis. Incorporating one or more unnatural amino acids may
comprise a tRNA/tRNA synthetase pair. The tRNA/tRNA synthetase pair
may comprise a tRNA/aminoacyl-tRNA synthetase pair. The tRNA/tRNA
synthetase pair may comprise a tRNATyr/tyrosyl-tRNA synthetase
pair.
[0014] Coupling L1'' to X may occur at the one or more unnatural
amino acids in X. Coupling L2'' to Y may occur at the one or more
unnatural amino acids in Y. Coupling L1'' to X and coupling L2'' to
Y may occur sequentially. Coupling L1'' to X and coupling L2 to Y
may occur simultaneously. Coupling L1'' to X may occur prior to
coupling L2'' to Y. Coupling L2'' to Y may occur prior to coupling
L1'' to X. Coupling L1'' to X and/or coupling L2'' to Y may
comprise forming one or more oxime bonds between L1'' and X and/or
between L2'' and Y. Coupling L1'' to X and/or coupling L2'' to Y
may comprise forming one or more stable bonds between L1'' and X
and/or between L2'' and Y. Coupling L1'' to X and/or coupling L2''
to Y may comprise forming one or more covalent bonds between L1''
and X and/or between L2'' and Y. Coupling L1'' to X and/or coupling
L2'' to Y may comprise forming one or more non-covalent bonds
between L1'' and X and/or between L2'' and Y. Coupling L1'' to X
and/or coupling L2'' to Y may comprise forming one or more ionic
bonds between L1'' and X and/or between L2'' and Y.
[0015] Linking the first intermediate to the second intermediate
may comprise a Huisgen-cycloaddition. Linking the first
intermediate to the second intermediate may comprise a Diels-Halder
reaction Linking the first intermediate to the second intermediate
may comprise a hetero Diels-Alder reaction Linking the first
intermediate to the second intermediate may comprise an
enzyme-mediated reaction Linking the first intermediate to the
second intermediate may comprise a copper-free [3+2]
Huisgen-cycloaddition reaction Linking the first intermediate to
the second intermediate may produce an oxime Linking the first
intermediate to the second intermediate may produce a tetrazole
Linking the first intermediate to the second intermediate may
produce a Diels Alder adduct Linking the first intermediate to the
second intermediate may produce a hetero Diels Alder adduct Linking
the first intermediate to the second intermediate may produce an
aromatic substitution reaction product Linking the first
intermediate to the second intermediate may produce a nucleophilic
substitution reaction product Linking the first intermediate to the
second intermediate may produce an ester Linking the first
intermediate to the second intermediate may produce an amide
Linking the first intermediate to the second intermediate may
produce a carbamate Linking the first intermediate to the second
intermediate may produce an ether Linking the first intermediate to
the second intermediate may produce a thioether Linking the first
intermediate to the second intermediate may produce a Michael
reaction product Linking the first intermediate to the second
intermediate may produce cycloaddition product Linking the first
intermediate to the second intermediate may produce a metathesis
reaction product Linking the first intermediate to the second
intermediate may produce a metal-mediated cross-coupling reaction
product Linking the first intermediate to the second intermediate
may produce a radical polymerization product Linking the first
intermediate to the second intermediate may produce an oxidative
coupling product Linking the first intermediate to the second
intermediate may produce an acyl-transfer reaction product Linking
the first intermediate to the second intermediate may produce a
photo click reaction product. Linking the first intermediate to the
second intermediate may produce a disulfide bridge Linking the
first intermediate to the second intermediate may produce a
maleimide bridge. The distance between X and Y may be less than or
equal to 50, 45, 40, 35, 30, 25, or 20 angstroms (.ANG.). The
distance between X and Y may be greater than or equal to 5
angstroms (.ANG.).
[0016] Further disclosed herein are pharmaceutical compositions
comprising a bispecific antibody, wherein the bispecific antibody
comprises an anti-CD3 antibody or anti-CD3 antibody fragment; a
second antibody or antibody fragment, wherein the anti-CD3 antibody
or anti-CD3 antibody fragment is site-specifically connected to the
second antibody or antibody fragment. The anti-CD3 antibody or
anti-CD3 antibody fragment may be site-specifically connected to
the second antibody or antibody fragment by one or more linkers.
The anti-CD3 antibody or anti-CD3 antibody fragment may comprise
one or more unnatural amino acids. The second antibody or antibody
fragment comprises one or more unnatural amino acids. The anti-CD3
antibody or anti-CD3 antibody fragment and the second antibody or
antibody fragment may comprise one or more unnatural amino acids.
The pharmaceutical composition may further comprise a
pharmaceutically acceptable diluent, a pharmaceutically acceptable
excipient or a pharmaceutically acceptable carrier.
[0017] Disclosed herein are methods for treating a disease or
condition in a subject in need thereof, comprising administering a
bispecific antibody, wherein the bispecific antibody comprises an
anti-CD3 antibody or anti-CD3 antibody fragment; a second antibody
or antibody fragment, wherein the anti-CD3 antibody or anti-CD3
antibody fragment is site-specifically connected to the second
antibody or antibody fragment. The anti-CD3 antibody or anti-CD3
antibody fragment may be site-specifically connected to the second
antibody or antibody fragment by one or more linkers. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise one or more
unnatural amino acids. The second antibody or antibody fragment
comprises one or more unnatural amino acids. The anti-CD3 antibody
or anti-CD3 antibody fragment and the second antibody or antibody
fragment may comprise one or more unnatural amino acids. The
disease or condition may be a cancer. The cancer may be a breast
cancer. The cancer may be a glioma or glioblastoma. The cancer may
be a multiple myeloma. The cancer may be an acute myeloid leukemia
(AML). The cancer may be selected from an acute lymphoblastic
leukemia (ALL), a B-cell chronic lymphocytic leukemia (B-CLL) and a
mantle cell lymphoma (MCL). The disease or condition may be a
pathogenic infection. The disease or condition may be an
inflammatory disease, an autoimmune disease or a metabolic disease.
The bispecific antibody or pharmaceutical composition may be
administered by parenteral administration. The parenteral
administration may comprise intravenous administration,
subcutaneous administration, intraperitoneal administration,
intramuscular administration, intravascular administration,
intrathecal administration, intravitreal administration, or
infusion. The bispecific antibody or pharmaceutical composition may
be administered by a microneedle device. The bispecific antibody or
pharmaceutical composition may be administered by topical, oral, or
nasal administration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The foregoing summary, as well as the following detailed
description of the disclosure, may be better understood when read
in conjunction with the appended figures. For the purpose of
illustrating the disclosure, shown in the figures are embodiments
which are presently preferred. It should be understood, however,
that the disclosure is not limited to the precise arrangements,
examples and instrumentalities shown.
[0019] The invention may be best understood from the following
detailed description when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to-scale. On the contrary,
the dimensions of the various features are arbitrarily expanded or
reduced for clarity. Included in the drawings are the following
figures.
[0020] FIG. 1 depicts a ribbon diagram of a UCHT1-Fab fragment.
[0021] FIG. 2A depicts the structure of p-acetylphenylaline.
[0022] FIG. 2B depicts the structure of bifunctional ethylene
glycol linkers
[0023] FIG. 2C depicts a general scheme for the generation of
bispecific antibodies
[0024] FIG. 3A-E depict ESI-MS analysis of Fab fragments before
linker and after linker conjugation. FIG. 3A depicts Herceptin Fab
mutant (LS202X, X=pAcF); FIG. 3B depicts Herceptin
Fab-(PEG).sub.4-Az; FIG. 3C depicts Herceptin
Fab-(PEG).sub.4-Octyne; FIG. 3D depicts UCHT1 Fab mutant (HK138X,
X=pAcF); and FIG. 3E depicts UCHT1 Fab-(PEG).sub.4-Octyne.
[0025] FIG. 3F-J depict deconvoluted mass spectrum of Fab fragments
before and after linker conjugation. FIG. 3F depicts Herceptin Fab
mutant (LS202X, X=pAcF); FIG. 3G depicts Herceptin
Fab-(PEG).sub.4-Az; FIG. 3H depicts Herceptin
Fab-(PEG).sub.4-Octyne; FIG. 3I depicts UCHT1 Fab mutant (HK138X,
X=pAcF); and FIG. 3J depicts UCHT1 Fab-(PEG).sub.4-Octyne.
[0026] FIG. 4A-B depict an SDS-PAGE gel (FIG. 4A) and size
exclusion chromatography FPLC trace (FIG. 4B) of the
anti-HER2/anti-CD3 heterodimer.
[0027] FIG. 5A-D depict bispecific binding of anti-HER2/anti-CD3
Fab heterodimer. FACS-based binding assay with Jurkat (CD3+) cells
(FIG. 5A) and SK-BR-3 (HER2+) cells (FIG. 5B). FIG. 5C-D depicts
fluorescence microscope images of the interaction between SK-BR3
cells (larger, dark grey circles, highlighted by the vertical
arrows in FIG. 5C) and Jurkat cells (smaller, light grey circles,
highlighted by the horizontal arrows in FIG. 5C-D) in the presence
of the conjugated anti-HER2/anti-CD3 heterodimer (FIG. 5C) or in
the presence of a mixture of unconjuaged anti-HER2 Fab and anti-CD3
Fab at a 1:1 ratio (FIG. 5D).
[0028] FIG. 6 depicts a graph of dose-dependent cytoxicity with
MDA-MB-435/HER2+ cells in the presence of human PBMCs and antibody
heterodimer.
[0029] FIG. 7A-D depict microscopic images from a cytoxicity assay.
FIG. 7A depicts microscopic images from a cytoxicity assay with
HER2- cells treated with a Fab mixture; FIG. 7B depicts microscopic
images from a cytoxicity assay with HER2- cells treated with a
bispecific antibody heterodimer; FIG. 7C depicts microscopic images
from a cytoxicity assay with HER2+ cells treated with a Fab
mixture; FIG. 7D depicts microscopic images from a cytoxicity assay
with HER2+ cells treated with a bispecific antibody
heterodimer.
[0030] FIG. 8 depicts growth curves of MDA-MB-435/HER2+ cells
premixed with hPBMC and Matrigel. The ratios of the cancer cells to
hPBMCs were 1:1 (square), 1:2 (triangle) and 1:5 (x).
[0031] FIG. 9 depicts a graph of the tumor volume of a
MDA-MB-435/HER2+ tumor treated with a bispecific antibody (circle),
Fab mixture (square) or PBS (triangle).
[0032] FIG. 10 depicts IVIS imaging of two heterodimer treated mice
(left two) and two unconjugated Fab treated mice (right two).
[0033] FIG. 11 depicts excised tumors or MatriGel from a
preventative anti-HER2/anti-CD3 Fab heterodimer study. The top row
is the heterodimer treated group (n=7). The middle row is the
unconjugated Fabs treated group (n=7). The bottom row is the
PBS-treated group (n=6).
[0034] FIG. 12 shows a general scheme for synthesizing bifunctional
linkers.
[0035] FIG. 13 depicts a graph of the cytoxicity of Ramos cells
treated with an anti-CD20/anti-CD3 bispecific antibody or treated
with unconjugated Fab fragments.
[0036] FIG. 14 depicts a graph of the cytoxicity of A549 cells
treated with an anti-EGFR/anti-CD3 bispecific antibody or treated
with unconjugated Fab fragments.
[0037] FIG. 15 depicts a graph of the cytoxicity of HT29 cells
treated with an anti-EGFR/anti-CD3 bispecific antibody or treated
with unconjugated Fab fragments.
[0038] FIG. 16 depicts a graph of the cytoxicity of AGS cells
treated with an anti-EGFR/anti-CD3 bispecific antibody or treated
with unconjugated Fab fragments.
[0039] FIG. 17A depticts the antigen binding fragments (Fab's) of
anti-CS1 (huLuc3) and anti-CD3 (UCHT1) antibodies that were
individually expressed in E. coli with pAcF suppressing a TAG
mutation (at Serine 202) purified and conjugated using the
optimized heterobifunctional linkers in FIG. 17B.
[0040] FIG. 17B depicts heterobifunctional linkers. The
Tet-TEG-ONH.sub.2 linker was conjugated to CS1 using the aminooxy
functionality of the linker and the ketone of the pAcF incorporated
at Serine 202 in an oxime ligation. The TCO-TEG-ONH.sub.2 was
conjugated to was conjugated to UCHT1 using the aminooxy
functionality of the linker and the ketone of pAcF incorporated at
Serine 202 in an oxime ligation. The two proteins were subsequently
"clicked" together to create the BiFab shown in FIG. 17A.
[0041] FIG. 17C shows the cytotoxicity of the BiFab, unconjugated
monomers, and unconjugated monomers mixed was assayed using freshly
purified, crude, human peripheral blood monocytes (PBMCs) against
MM.1S cells in an overnight assay. Cytotoxicity results were
readout by an LDH assay following standard protocols.
[0042] FIG. 18 depicts exemplary linkers.
[0043] FIG. 19A shows masses of CS1 Fab S202pAcF and UCHT1 Fab
S202pAcF prior to conjugation to their respective linkers.
[0044] FIG. 19B shows masses of CS1 Fab S202pAcF and UCHT1 Fab
S202pAcF conjugated to the Tet-TEG-ONH2 (tet) and TCO-Tet-ONH2
(TCO) linkers respectively.
[0045] FIG. 19C shows the mass of the CS1.times.UCHT1 biFab.
[0046] FIG. 20A shows selective binding of anti CLL-1/anti-CD3
biFab to CLL-1+HL-60 cells.
[0047] FIG. 20B shows selective binding of anti-CD33/anti-CD3 biFab
to CD33+HL-60 cells.
[0048] FIG. 21A shows cytotoxicity of anti-CLL-1/anti-CD3 biFab
towards CLL-1+HL-60 cells.
[0049] FIG. 21B shows cytotoxicity of anti-CD33/anti-CD3 biFab
towards CD33+HL-60 cells.
[0050] FIG. 22A shows HL-60 IFN gamma release due to
anti-CD33/anti-CD3 biFab or anti-CLL-1/anti-CD3 biFab.
[0051] FIG. 22B shows HL-60 IL-2 release due to anti-CD33/anti-CD3
biFab or anti-CLL-1/anti-CD3 biFab.
[0052] FIG. 23 shows expression and purification of
UCHT1/anti-EGFRvIII biFabs. Antigen binding fragments (Fab's) of
anti-EGFR/EGFRvIII (hu806) and anti-CD3 (UCHT1) antibodies were
individually expressed in E. coli with pAcF suppressing a TAG
mutation (at Serine 202 LC, hu806 or Lysine 138 HC, UCHT1) purified
and conjugated using optimized heterobifunctional linkers.
[0053] FIG. 24A shows masses of Hu806 Fab S202pAcF and UCHT1 Fab
S202pAcF prior to conjugation to their respective linkers.
[0054] FIG. 24B shows masses of Hu806 Fab S202pAcF and UCHT1 Fab
S202pAcF conjugated to the Tet-TEG-ONH2 and TCO-Tet-ONH2 linkers
respectively.
[0055] FIG. 24C shows the mass of the Hu806-UCHT1 BiFab.
[0056] FIG. 25 shows cytotoxicity of the Hu806-UCHT1 BiFab,
unconjugated monomers, and unconjugated monomers mixed was assayed
using freshly purified activated human peripheral blood monocytes
(PBMCs) against A431 cells in an overnight assay. Cytotoxicity
results were readout by an LDH assay following standard
protocols.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to
particular method or composition described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting. Examples are put forth so as to
provide those of ordinary skill in the art with a complete
disclosure and description of how to make and use the present
invention, and are not intended to limit the scope of what the
inventors regard as their invention nor are they intended to
represent that the experiments below are all or the only
experiments performed. Efforts have been made to ensure accuracy
with respect to numbers used (e.g. amounts, temperature, etc.) but
some experimental errors and deviations should be accounted for.
Unless indicated otherwise, parts are parts by weight, molecular
weight is weight average molecular weight, temperature is in
degrees Centigrade, and pressure is at or near atmospheric.
[0058] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0059] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein may be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supersedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0060] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method may be carried out in the order of events recited or in any
other order which is logically possible.
[0061] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the peptide" includes reference to one or more
peptides and equivalents thereof, e.g. polypeptides, known to those
skilled in the art, and so forth.
[0062] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0063] Disclosed herein are immunoglobulin constructs or bispecific
antibodies. As used herein, the terms "immunoglobulin",
"immunoglobulin construct" and "bispecific antibody" are used
interchangeably. Generally, the bispecific antibody comprises one
or more unnatural amino acids. The bispecific antibody may comprise
an anti-CD3 antibody or anti-CD3 antibody fragment and a second
antibody or antibody fragment, wherein the anti-CD3 antibody or
anti-CD3 antibody fragment and the second antibody or antibody
fragment are site-specifically connected.
[0064] The bispecific antibody may comprise an anti-CD3 antibody or
anti-CD3 antibody fragment and an anti-EGFRvIII antibody or
antibody fragment, wherein the anti-CD3 antibody or anti-CD3
antibody fragment and the anti-EGFRvIII antibody or antibody
fragment are site-specifically connected. The anti-CD3 antibody or
anti-CD3 antibody fragment and/or the anti-EGFRvIII antibody or
antibody fragment may comprise one or more unnatural amino
acids.
[0065] The bispecific antibody may comprise an anti-CD3 antibody or
anti-CD3 antibody fragment and an anti-Her2 antibody or antibody
fragment, wherein the anti-CD3 antibody or anti-CD3 antibody
fragment and the anti-Her2 antibody or antibody fragment are
site-specifically connected. The anti-CD3 antibody or anti-CD3
antibody fragment and/or the anti-Her2 antibody or antibody
fragment may comprise one or more unnatural amino acids.
[0066] The bispecific antibody may comprise an anti-CD3 antibody or
anti-CD3 antibody fragment and an anti-CS-1 antibody or antibody
fragment, wherein the anti-CD3 antibody or anti-CD3 antibody
fragment and the anti-CS-1 antibody or antibody fragment are
site-specifically connected. The anti-CD3 antibody or anti-CD3
antibody fragment and/or the anti-CS-1 antibody or antibody
fragment may comprise one or more unnatural amino acids.
[0067] The bispecific antibody may comprise an anti-CD3 antibody or
anti-CD3 antibody fragment and an anti-CLL-1 antibody or antibody
fragment, wherein the anti-CD3 antibody or anti-CD3 antibody
fragment and the anti-CLL-1 antibody or antibody fragment are
site-specifically connected. The anti-CD3 antibody or anti-CD3
antibody fragment and/or the anti-CLL-1 antibody or antibody
fragment may comprise one or more unnatural amino acids.
[0068] The bispecific antibody may comprise an anti-CD3 antibody or
anti-CD3 antibody fragment and an anti-CD33 antibody or antibody
fragment, wherein the anti-CD3 antibody or anti-CD3 antibody
fragment and the anti-CD33 antibody or antibody fragment are
site-specifically connected. The anti-CD3 antibody or anti-CD3
antibody fragment and/or the anti-CD33 antibody or antibody
fragment may comprise one or more unnatural amino acids.
[0069] Further disclosed herein is a bispecific antibody of Formula
I: X-L1-Y or Formula IA: Y-L1-X, wherein (a) X comprises at least a
portion of an anti-CD3 antibody or anti-CD3 antibody fragment; (b)
L1 is a linker; and (c) Y comprises at least a portion of a second
antibody or antibody fragment, wherein X is site-specifically
linked to Y by the linker. X may comprise one or more unnatural
amino acids. Y may comprise one or more unnatural amino acids. X
and Y may comprise one or more unnatural amino acids.
[0070] Further disclosed herein is a bispecific antibody of Formula
II: X-L1-L2-Y or an antibody of Formula IIA: Y-L2-L1-X, wherein (a)
X comprises at least a portion of an anti-CD3 antibody or anti-CD3
antibody fragment; (b) L1 and L2 are linkers; and (c) Y comprises
at least a portion of a second antibody or antibody fragment,
wherein X is site-specifically linked to Y by the linkers. X may
comprise one or more unnatural amino acids. Y may comprise one or
more unnatural amino acids. X and Y may comprise one or more
unnatural amino acids.
I. Antibodies and Antibody Fragments
[0071] The bispecific antibodies disclosed herein (e.g., an
antibody of Formula I, Formula IA, Formula II, Formula IIA) may
bind to two or more different antigens. The bispecific antibody may
comprise an antibody, antibody fragment, or combination thereof.
The bispecific antibody may comprise an
immunoglobulin-immunoglobulin (Ig-Ig) construct, wherein X
comprises an immunoglobulin and Y comprises an immunoglobulin. The
bispecific antibody may comprise an immunoglobulin-Fab (Ig-Fab)
construct, wherein X comprises an immunoglobulin and Y comprises a
Fab fragment. The bispecific antibody may comprise a Fab-Fab
construct, wherein X comprises a Fab fragment and Y comprises a Fab
fragment. As depicted by Formula I, IA, II, and HA, X and Y may be
linked by one or more linkers (e.g., L1, L2).
[0072] As used herein, the term "antibody fragment" may refer to
any form of an antibody other than the full-length form. Antibody
fragments herein include antibodies that are smaller components
that exist within full-length antibodies, and antibodies that have
been engineered. Antibody fragments include, but are not limited
to, Fv, Fc, Fab, and (Fab')2, single chain Fv (scFv), diabodies,
triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1,
CDR2, CDR3, combinations of CDR's, variable regions, framework
regions, constant regions, heavy chains, light chains, alternative
scaffold non-antibody molecules, and bispecific antibodies. Unless
specifically noted otherwise, statements and claims that use the
term "antibody" or "antibodies" may specifically include "antibody
fragment" and "antibody fragments."
[0073] The antibodies disclosed herein may be human, fully human,
humanized, human engineered, non-human, and/or chimeric. For
example, the antibody of Formula I may be a humanized antibody. In
another example, the antibody of Formula II is a chimeric antibody.
The antibodies disclosed herein may be based on or derived from
human, fully human, humanized, human engineered, non-human and/or
chimeric antibodies. For example, X and/or Y of Formula IA may be
based on or derived from a human engineered antibody.
Alternatively, X and/or Y of Formula IIA may be based on or derived
from a non-human antibody. Non-human antibody may be humanized to
reduce immunogenicity to humans, while retaining the specificity
and affinity of the parental non-human antibody. Generally, a
humanized antibody comprises one or more variable domains in which
CDRs (or portions thereof) are derived from a non-human antibody,
and FRs (or portions thereof) are derived from human antibody
sequences. A humanized antibody optionally also comprises at least
a portion of a human constant region. In some embodiments, some FR
residues in a humanized antibody are substituted with corresponding
residues from a non-human antibody (e.g., the antibody from which
the CDR residues are derived), e.g., to restore or improve antibody
specificity or affinity.
[0074] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua
et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005); Klimka et al., Br. J.
Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR shuffling); and Studnicka et al., U.S. Pat. No.
5,766,886.
[0075] Chimeric antibodies may refer to antibodies created through
the joining of two or more antibody genes which originally encoded
for separate antibodies. A chimeric antibody may comprise at least
one amino acid from a first antibody and at least one amino acid
from a second antibody, wherein the first and second antibodies are
different. The antibodies disclosed herein may comprise antibody
sequences from two or more different antibodies. For example, X of
the antibodies disclosed herein (e.g., antibody of Formula I, IA,
II or HA) may be from a first antibody and Y of the antibodies
disclosed herein (e.g., antibody of Formula I, IA, II or IIA) may
be from a second antibody, wherein the first and second antibodies
are different. In another example, X of the antibodies disclosed
herein (e.g., antibody of Formula I, IA, II or HA) may be a
chimeric antibody. Alternatively, or additionally, Y of the
antibodies disclosed herein may be a chimeric antibody. The two or
more different antibodies may be from the same species. For
example, the species may be a bovine species, human species, or
murine species. The two or more different antibodies may be from
the same type of animal. For example the two or more different
antibodies may be from a cow. The two or more different antibodies
may be from a human. Alternatively, the two or more different
antibodies are from different species. For example, the two or more
different antibodies are from a human species and bovine species.
In another example, the two or more different antibodies are from a
bovine species and a non-bovine species. In another example, the
two or more different antibodies are from a human species and a
non-human species.
[0076] The anti-CD3 antibody or anti-CD3 antibody fragment may
comprise at least a portion of a sequence selected from SEQ ID NOs:
1-2. The anti-CD3 antibody or anti-CD3 antibody fragment may
comprise a sequence that is at least 50% identical to a sequence
selected from SEQ ID NOs: 1-2. The anti-CD3 antibody or anti-CD3
antibody fragment may comprise a sequence that is at least 60%
identical to a sequence selected from SEQ ID NOs: 1-2. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise a sequence that
is at least 70% identical to a sequence selected from SEQ ID NOs:
1-2. The anti-CD3 antibody or anti-CD3 antibody fragment may
comprise a sequence that is at least 80% identical to a sequence
selected from SEQ ID NOs: 1-2. The anti-CD3 antibody or anti-CD3
antibody fragment may comprise a sequence that is at least 50%
identical to a sequence selected from SEQ ID NOs: 1-2. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise a sequence that
is at least 85% identical to a sequence selected from SEQ ID NOs:
1-2. The anti-CD3 antibody or anti-CD3 antibody fragment may
comprise a sequence that is at least 90% identical to a sequence
selected from SEQ ID NOs: 1-2. The anti-CD3 antibody or anti-CD3
antibody fragment may comprise a sequence that is at least 95%
identical to a sequence selected from SEQ ID NOs: 1-2. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise a sequence that
is at least 97% identical to a sequence selected from SEQ ID NOs:
1-2.
[0077] The anti-CD3 antibody or anti-CD3 antibody fragment may
comprise a sequence comprising five or more amino acids based on or
derived from a sequence selected from SEQ ID NOs: 1-2. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise a sequence
comprising 6, 7, 8, 9, 10 or more amino acids based on or derived
from a sequence selected from SEQ ID NOs: 1-2. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise a sequence
comprising 15, 16, 17, 18, 19, 20 or more amino acids based on or
derived from a sequence selected from SEQ ID NOs: 1-2. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise a sequence
comprising 25, 30, 35, 40, 45, 50 or more amino acids based on or
derived from a sequence selected from SEQ ID NOs: 1-2. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise a sequence
comprising 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino
acids based on or derived from a sequence selected from SEQ ID NOs:
1-2. The amino acids may be consecutive. The amino acids may be
non-consecutive.
[0078] The second antibody or antibody fragment may comprise at
least a portion of a sequence selected from SEQ ID NOs: 3-16. The
second antibody or antibody fragment may comprise a sequence that
is at least 50% identical to a sequence selected from SEQ ID NOs:
3-16. The second antibody or antibody fragment may comprise a
sequence that is at least 60% identical to a sequence selected from
SEQ ID NOs: 3-16. The second antibody or antibody fragment may
comprise a sequence that is at least 70% identical to a sequence
selected from SEQ ID NOs: 3-16. The second antibody or antibody
fragment may comprise a sequence that is at least 80% identical to
a sequence selected from SEQ ID NOs: 3-16. The second antibody or
antibody fragment may comprise a sequence that is at least 50%
identical to a sequence selected from SEQ ID NOs: 3-16. The second
antibody or antibody fragment may comprise a sequence that is at
least 85% identical to a sequence selected from SEQ ID NOs: 3-16.
The second antibody or antibody fragment may comprise a sequence
that is at least 90% identical to a sequence selected from SEQ ID
NOs: 3-16. The second antibody or antibody fragment may comprise a
sequence that is at least 95% identical to a sequence selected from
SEQ ID NOs: 3-16. The second antibody or antibody fragment may
comprise a sequence that is at least 97% identical to a sequence
selected from SEQ ID NOs: 3-16.
[0079] The second antibody or antibody fragment may comprise a
sequence comprising five or more amino acids based on or derived
from a sequence selected from SEQ ID NOs: 3-16. The second antibody
or antibody fragment may comprise a sequence comprising 6, 7, 8, 9,
10 or more amino acids based on or derived from a sequence selected
from SEQ ID NOs: 3-16. The second antibody or antibody fragment may
comprise a sequence comprising 15, 16, 17, 18, 19, 20 or more amino
acids based on or derived from a sequence selected from SEQ ID NOs:
3-16. The second antibody or antibody fragment may comprise a
sequence comprising 25, 30, 35, 40, 45, 50 or more amino acids
based on or derived from a sequence selected from SEQ ID NOs: 3-16.
The second antibody or antibody fragment may comprise a sequence
comprising 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino
acids based on or derived from a sequence selected from SEQ ID NOs:
3-16. The amino acids may be consecutive. The amino acids may be
non-consecutive.
[0080] The antibodies disclosed herein may be based on or derived
from an antibody or antibody fragment from a mammal, bird, fish,
amphibian or reptile. Mammals include, but are not limited to,
carnivores, rodents, elephants, marsupials, rabbits, bats,
primates, seals, anteaters, cetaceans, odd-toed ungulates and
even-toed ungulates. The mammal may be a human, non-human primate,
mouse, sheep, cat, dog, cow, horse, goat, or pig. The mammal may be
a human. The mammal may be a cynomolgus monkey.
[0081] Birds include, but are not limited to, albatrosses,
hummingbirds, eagles, ostriches, cardinals, kiwis, and penguins.
Fish may be cartilaginous fishes, ray-finned fishes, or lobe-fined
fishes. Amphibians may include, but are not limited to, newts,
salamanders, frogs and toads. Examples of reptiles include, but are
not limited to, turtles, squamates, crocodiles and tuataras.
Squamates may include amphisbaenians, lizards and snakes.
IA. Antibody or Antibody Fragments of X
[0082] The bispecific antibodies of Formulas I, IA, II, and/or IIA
disclosed herein may comprise X, wherein X comprises at least a
portion of an anti-CD3 antibody or anti-CD3 antibody fragment
comprising one or more unnatural amino acids. X may comprise an
entire anti-CD3 antibody. The anti-CD3 antibody may be UCHT1. X may
comprise at least a portion of an anti-CD3 antibody. X may comprise
at least a portion of a monoclonal anti-CD3 antibody. X may
comprise at least a portion of a CD3 polyclonal antibody. X may
comprise at least a portion of a multivalent anti-CD3 antibody.
[0083] X may comprise an anti-CD3 antibody. X may comprise at least
a portion of a Fab fragment of an anti-CD3 antibody. X may comprise
an antibody fragment of an anti-CD3 antibody. X may comprise at
least a portion of an anti-CD3 antibody. The portion of the
antibody may comprise an antibody fragment. Antibody fragments
include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single
chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional
hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDR's,
variable regions, framework regions, constant regions, heavy
chains, light chains, alternative scaffold non-antibody molecules,
and bispecific antibodies. X may comprise at least a portion of a
heavy chain (HC) of an anti-CD3 antibody. X may comprise at least a
portion of a light chain (LC) of a anti-CD3 antibody. X may
comprise at least a portion of a variable region of an anti-CD3
antibody. X may comprise at least a portion of a constant region of
an anti-CD3 antibody. X may comprise an antibody or antibody
fragment that binds to a portion of CD3. The portion of CD3 may
comprise a CD3 gamma chain or a portion thereof. The portion of CD3
may comprise a CD3 delta chain or a portion thereof. The portion of
CD3 may comprise a CD3 epsilon chain or a portion thereof. The
portion of CD3 may comprise a combination of a CD3 gamma chain, a
CD3 delta chain and a CD3 epsilon chain.
[0084] X may comprise at least a portion of a sequence selected
from SEQ ID NOs: 1-2. X may comprise a sequence that is at least
50% identical to a sequence selected from SEQ ID NOs: 1-2. X may
comprise a sequence that is at least 60% identical to a sequence
selected from SEQ ID NOs: 1-2. X may comprise a sequence that is at
least 70% identical to a sequence selected from SEQ ID NOs: 1-2. X
may comprise a sequence that is at least 80% identical to a
sequence selected from SEQ ID NOs: 1-2. X may comprise a sequence
that is at least 50% identical to a sequence selected from SEQ ID
NOs: 1-2. X may comprise a sequence that is at least 85% identical
to a sequence selected from SEQ ID NOs: 1-2. X may comprise a
sequence that is at least 90% identical to a sequence selected from
SEQ ID NOs: 1-2. X may comprise a sequence that is at least 95%
identical to a sequence selected from SEQ ID NOs: 1-2. X may
comprise a sequence that is at least 97% identical to a sequence
selected from SEQ ID NOs: 1-2.
[0085] X may comprise a sequence comprising five or more amino
acids based on or derived from a sequence selected from SEQ ID NOs:
1-2. X may comprise a sequence comprising 6, 7, 8, 9, 10 or more
amino acids based on or derived from a sequence selected from SEQ
ID NOs: 1-2. X may comprise a sequence comprising 15, 16, 17, 18,
19, 20 or more amino acids based on or derived from a sequence
selected from SEQ ID NOs: 1-2. X may comprise a sequence comprising
25, 30, 35, 40, 45, 50 or more amino acids based on or derived from
a sequence selected from SEQ ID NOs: 1-2. X may comprise a sequence
comprising 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino
acids based on or derived from a sequence selected from SEQ ID NOs:
1-2. The amino acids may be consecutive. The amino acids may be
non-consecutive.
[0086] X may comprise an antibody or at least a portion of an
antibody that is a human, fully human, humanized, human engineered,
non-human, or chimeric antibody. X may comprise an antibody or at
least a portion of an antibody that is a mammalian antibody. X may
comprise an antibody or at least a portion of an antibody that is a
non-mammalian antibody. X may comprise an antibody or at least a
portion of an antibody that is cross-reactive. X may comprise an
antibody or at least a portion of an antibody that is species
cross-reactive with human and cynomolgus monkey.
[0087] X may comprise a sequence based on or derived from one or
more anti-CD3 antibody and/or anti-CD3 antibody fragment sequences.
X may comprise a sequence that is at least about 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more
homologous to a sequence based on or derived from one or more
anti-CD3 antibodies and/or anti-CD3 antibody fragments. X may
comprise a sequence that is at least about 70% homologous to a
sequence based on or derived from one or more anti-CD3 antibodies
and/or anti-CD3 antibody fragments. X may comprise a sequence that
is at least about 80% homologous to a sequence based on or derived
from one or more anti-CD3 antibodies and/or anti-CD3 antibody
fragments. X may comprise a sequence that is at least about 90%
homologous to a sequence based on or derived from one or more
anti-CD3 antibodies and/or anti-CD3 antibody fragments. X may
comprise a sequence that is at least about 95% homologous to a
sequence based on or derived from one or more anti-CD3 antibodies
and/or anti-CD3 antibody fragments. The sequence may be a peptide
sequence. Alternatively, the sequence is a nucleotide sequence.
[0088] X may comprise a peptide sequence that differs from a
peptide sequence based on or derived from one or more anti-CD3
antibodies and/or anti-CD3 antibody fragments by less than or equal
to about 20, 17, 15, 12, 10, 8, 6, 5, 4 or fewer amino acids. X may
comprise a peptide sequence that differs from a peptide sequence
based on or derived from one or more anti-CD3 antibodies and/or
anti-CD3 antibody fragments by less than or equal to about 4 or
fewer amino acids. X may comprise a peptide sequence that differs
from a peptide sequence based on or derived from one or more
anti-CD3 antibodies and/or anti-CD3 antibody fragments by less than
or equal to about 3 or fewer amino acids. X may comprise a peptide
sequence that differs from a peptide sequence based on or derived
from one or more anti-CD3 antibodies and/or anti-CD3 antibody
fragments by less than or equal to about 2 or fewer amino acids. X
may comprise a peptide sequence that differs from a peptide
sequence based on or derived from one or more anti-CD3 antibodies
and/or anti-CD3 antibody fragments by less than or equal to about 1
or fewer amino acids. The amino acids may be consecutive,
nonconsecutive, or a combination thereof. For example, X may
comprise a peptide sequence that differs from a peptide sequence
based on or derived from one or more anti-CD3 antibodies and/or
anti-CD3 antibody fragments by less than about 3 consecutive amino
acids. Alternatively, or additionally, X may comprise a peptide
sequence that differs from a peptide sequence based on or derived
from one or more anti-CD3 antibodies and/or anti-CD3 antibody
fragments by less than about 2 non-consecutive amino acids. In
another example, X may comprise a peptide sequence that differs
from a peptide sequence based on or derived from one or more
anti-CD3 antibodies and/or anti-CD3 antibody fragments by less than
about 5 amino acids, wherein 2 of the amino acids are consecutive
and 2 of the amino acids are non-consecutive.
[0089] X may be encoded by a nucleotide sequence that differs from
a nucleotide sequence based on or derived from one or more anti-CD3
antibodies and/or anti-CD3 antibody fragments by less than or equal
to about 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
7, 6, 5, 4 or fewer nucleotides or base pairs. X may be encoded by
a nucleotide sequence that differs from a nucleotide sequence based
on or derived from one or more CD3 antibodies and/or anti-CD3
antibody fragments by less than or equal to about 15 or fewer
nucleotides or base pairs. X may be encoded by a nucleotide
sequence that differs from a nucleotide sequence based on or
derived from one or more anti-CD3 antibodies and/or anti-CD3
antibody fragments by less than or equal to about 12 or fewer
nucleotides or base pairs. X may be encoded by a nucleotide
sequence that differs from a nucleotide sequence based on or
derived from one or more anti-CD3 antibodies and/or CD3 antibody
fragments by less than or equal to about 9 or fewer nucleotides or
base pairs. X may be encoded by a nucleotide sequence that differs
from a nucleotide sequence based on or derived from one or more
anti-CD3 antibodies and/or anti-CD3 antibody fragments by less than
or equal to about 6 or fewer nucleotides or base pairs. X may be
encoded by a nucleotide sequence that differs from a nucleotide
sequence based on or derived from one or more anti-CD3 antibodies
and/or anti-CD3 antibody fragments by less than or equal to about 4
or fewer nucleotides or base pairs. X may be encoded by a
nucleotide sequence that differs from a nucleotide sequence based
on or derived from one or more anti-CD3 antibodies and/or anti-CD3
antibody fragments by less than or equal to about 3 or fewer
nucleotides or base pairs. X may be encoded by a nucleotide
sequence that differs from a nucleotide sequence based on or
derived from one or more anti-CD3 antibodies and/or anti-CD3
antibody fragments by less than or equal to about 2 or fewer
nucleotides or base pairs. X may be encoded by a nucleotide
sequence that differs from a nucleotide sequence based on or
derived from one or more anti-CD3 antibodies and/or anti-CD3
antibody fragments by less than or equal to about 1 or fewer
nucleotides or base pairs. The nucleotides or base pairs may be
consecutive, nonconsecutive, or a combination thereof. For example,
X may be encoded by a nucleotide sequence that differs from a
nucleotide sequence based on or derived from one or more anti-CD3
antibodies and/or anti-CD3 antibody fragments by less than about 3
consecutive nucleotides or base pairs. Alternatively, or
additionally, X may be encoded by a nucleotide sequence that
differs from a nucleotide sequence based on or derived from one or
more anti-CD3 antibodies and/or anti-CD3 antibody fragments by less
than about 2 non-consecutive nucleotides or base pairs. In another
example, X may be encoded by a nucleotide sequence that differs
from a nucleotide sequence based on or derived from one or more
anti-CD3 antibodies and/or anti-CD3 antibody fragments by less than
about 5 nucleotides or base pairs, wherein 2 of the nucleotides or
base pairs are consecutive and 2 of the nucleotides or base pairs
are non-consecutive.
[0090] The peptide sequence of X may differ from the peptide
sequence of the anti-CD3 antibody or anti-CD3 antibody fragment
that it is based on and/or derived from by one or more amino acid
substitutions. The peptide sequence of X may differ from the
peptide sequence of the anti-CD3 antibody or anti-CD3 antibody
fragment that it is based on and/or derived from by two or more
amino acid substitutions. The peptide sequence of X may differ from
the peptide sequence of the anti-CD3 antibody or anti-CD3 antibody
fragment that it is based on and/or derived from by three or more
amino acid substitutions. The peptide sequence of X may differ from
the peptide sequence of the anti-CD3 antibody or anti-CD3 antibody
fragment that it is based on and/or derived from by four or more
amino acid substitutions. The peptide sequence of X may differ from
the peptide sequence of the anti-CD3 antibody or anti-CD3 antibody
fragment that it is based on and/or derived from by five or more
amino acid substitutions. The peptide sequence of X may differ from
the peptide sequence of the anti-CD3 antibody or anti-CD3 antibody
fragment that it is based on and/or derived from by six or more
amino acid substitutions. The peptide sequence of X may differ from
the peptide sequence of the anti-CD3 antibody or anti-CD3 antibody
fragment that it is based on and/or derived from by 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 25 or more amino acid
substitutions.
[0091] The nucleotide sequence of X may differ from the nucleotide
sequence of the anti-CD3 antibody or anti-CD3 antibody fragment
that it is based on and/or derived from by one or more nucleotide
and/or base pair substitutions. The nucleotide sequence of X may
differ from the nucleotide sequence of the anti-CD3 antibody or
anti-CD3 antibody fragment that it is based on and/or derived from
by two or more nucleotide and/or base pair substitutions. The
nucleotide sequence of X may differ from the nucleotide sequence of
the anti-CD3 antibody or anti-CD3 antibody fragment that it is
based on and/or derived from by three or more nucleotide and/or
base pair substitutions. The nucleotide sequence of X may differ
from the nucleotide sequence of the anti-CD3 antibody or anti-CD3
antibody fragment that it is based on and/or derived from by four
or more nucleotide and/or base pair substitutions. The nucleotide
sequence of X may differ from the nucleotide sequence of the
anti-CD3 antibody or anti-CD3 antibody fragment that it is based on
and/or derived from by five or more nucleotide and/or base pair
substitutions. The nucleotide sequence of X may differ from the
nucleotide sequence of the anti-CD3 antibody or anti-CD3 antibody
fragment that it is based on and/or derived from by six or more
nucleotide and/or base pair substitutions. The nucleotide sequence
of X may differ from the nucleotide sequence of the anti-CD3
antibody or anti-CD3 antibody fragment that it is based on and/or
derived from by nine or more nucleotide and/or base pair
substitutions. The nucleotide sequence of X may differ from the
nucleotide sequence of the anti-CD3 antibody or anti-CD3 antibody
fragment that it is based on and/or derived from by twelve or more
nucleotide and/or base pair substitutions. The nucleotide sequence
of X may differ from the nucleotide sequence of the anti-CD3
antibody or anti-CD3 antibody fragment that it is based on and/or
derived from by fifteen or more nucleotide and/or base pair
substitutions. The nucleotide sequence of X may differ from the
nucleotide sequence of the anti-CD3 antibody or anti-CD3 antibody
fragment that it is based on and/or derived from by eighteen or
more nucleotide and/or base pair substitutions. The nucleotide
sequence of X may differ from the nucleotide sequence of the
anti-CD3 antibody or anti-CD3 antibody fragment that it is based on
and/or derived from by 20, 22, 24, 25, 27, 30 or more nucleotide
and/or base pair substitutions.
[0092] X may comprise one or more unnatural amino acids. X may
comprise two or more unnatural amino acids. X may comprise three or
more unnatural amino acids. X may comprise four or more unnatural
amino acids. X may comprise 5, 6, 7, 8, 9, 10 or more unnatural
amino acids.
[0093] X may be coupled to one or more linkers. X may be linked to
Y by one or more linkers. X may be linked to Y by two or more
linkers. X may be linked to Y by three or more linkers.
IB. Antibody and Antibody Fragments of Y
[0094] The bispecific antibodies of Formulas I, IA, II, and/or IIA
disclosed herein may comprise Y, wherein Y comprises at least a
portion of an antibody or antibody fragment. Y may comprise an
entire antibody. Y may comprise at least a portion of antibody. Y
may comprise at least a portion of a monoclonal antibody. Y may
comprise at least a portion of a polyclonal antibody. Y may
comprise at least a portion of a multivalent antibody.
[0095] Y may comprise at least a portion of an antibody. The
portion of the antibody may comprise an antibody fragment. Antibody
fragments include, but are not limited to, Fv, Fc, Fab, and
(Fab')2, single chain Fv (scFv), diabodies, triabodies,
tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3,
combinations of CDR's, variable regions, framework regions,
constant regions, heavy chains, light chains, alternative scaffold
non-antibody molecules, and bispecific antibodies. Y may comprise a
Fab fragment. Y may comprise at least a portion of a heavy chain
(HC) of an antibody. Y may comprise at least a portion of a light
chain (LC) of an antibody. Y may comprise at least a portion of a
variable region of an antibody. Y may comprise at least a portion
of a constant region of an antibody.
[0096] Y may comprise at least a portion of a sequence selected
from SEQ ID NOs: 3-16. Y may comprise a sequence that is at least
50% identical to a sequence selected from SEQ ID NOs: 3-16. Y may
comprise a sequence that is at least 60% identical to a sequence
selected from SEQ ID NOs: 3-16. Y may comprise a sequence that is
at least 70% identical to a sequence selected from SEQ ID NOs:
3-16. Y may comprise a sequence that is at least 80% identical to a
sequence selected from SEQ ID NOs: 3-16. Y may comprise a sequence
that is at least 50% identical to a sequence selected from SEQ ID
NOs: 3-16. Y may comprise a sequence that is at least 85% identical
to a sequence selected from SEQ ID NOs: 3-16. Y may comprise a
sequence that is at least 90% identical to a sequence selected from
SEQ ID NOs: 3-16. Y may comprise a sequence that is at least 95%
identical to a sequence selected from SEQ ID NOs: 3-16. Y may
comprise a sequence that is at least 97% identical to a sequence
selected from SEQ ID NOs: 3-16.
[0097] Y may comprise a sequence comprising five or more amino
acids based on or derived from a sequence selected from SEQ ID NOs:
3-16. Y may comprise a sequence comprising 6, 7, 8, 9, 10 or more
amino acids based on or derived from a sequence selected from SEQ
ID NOs: 3-16. Y may comprise a sequence comprising 15, 16, 17, 18,
19, 20 or more amino acids based on or derived from a sequence
selected from SEQ ID NOs: 3-16. Y may comprise a sequence
comprising 25, 30, 35, 40, 45, 50 or more amino acids based on or
derived from a sequence selected from SEQ ID NOs: 3-16. Y may
comprise a sequence comprising 55, 60, 65, 70, 75, 80, 85, 90, 95,
100 or more amino acids based on or derived from a sequence
selected from SEQ ID NOs: 3-16. The amino acids may be consecutive.
The amino acids may be non-consecutive.
[0098] Y may comprise at least a portion of an antibody. The
antibody may be an anti-CD20 antibody. The antibody may be an
anti-EGFR antibody. The anti-EGFR antibody may be an anti-Her1
antibody. The anti-EGFR antibody may be an anti-Her2 antibody. The
anti-EGFR antibody may be an anti-Her3 antibody. The anti-EGFR
antibody may be an anti-Her4 antibody. The antibody may be an
anti-EGFR antibody wherein the anti-EGFR antibody specifically
binds a mutated EGFR. The mutated EGFR may be EGFRvIII.
[0099] The antibody may bind a cell adhesion molecule. The antibody
may be an anti-CS1 antibody. The anti-CS1 antibody may comprise
elotozumab, also referred to as HuLuc63. The anti-CS1 antibody may
be humanized. The antibody may bind a receptor. The antibody may be
an anti-CLL-1 antibody. The antibody may be an anti-CD33 antibody.
The antibody may be an anti-ROR antibody. The ROR antibody may be
an anti-ROR1 antibody. The antibody may be an anti-CD44 antibody.
The anti-CD44 antibody may be an anti-CD44v6 antibody. The antibody
may be an anti-PVRL4 antibody. The antibody may be an
anti-interleukin receptor (ILR) antibody. The interleukin receptor
antibody may bind to an ILR selected from an IL1R, an IL2R, an
IL3R, an IL4R, an IL5R, an IL6R, an IL7R, an IL8R, an IL9R, an
IL10R, an IL11R, an IL12R, an IL13R, an IL14R, an IL15R and an
IL17R. The interleukin receptor antibody may bind to IL13R.alpha.2.
The interleukin receptor antibody may be an anti-IL13R.alpha.2
antibody.
[0100] The antibody may be a bispecific single chain (bsc)
antibody. The bsc antibody may be a bscWue-1 antibody.
[0101] Y may comprise a Fab fragment of an antibody. Y may comprise
a Fab fragment of an anti-B cell receptor antibody. Y may comprise
a Fab fragment of an anti-CD20 antibody. Y may comprise a Fab
fragment of an anti-EGFR antibody. Y may comprise a Fab fragment of
an anti-Her2 antibody. Y may comprise a Fab fragment of an
anti-EGFRvIII antibody. Y may comprise a Fab fragment of an
anti-CS1 antibody. Y may comprise a Fab fragment of an anti-CLL-1
antibody. Y may comprise a Fab fragment of an anti-CD33 antibody. Y
may comprise a Fab fragment of an anti-ROR1 antibody. Y may
comprise a Fab fragment of an anti-CD44v6 antibody. Y may comprise
a Fab fragment of an anti-PVRL4 antibody. Y may comprise a Fab
fragment of an anti-IL13R.alpha.2 antibody.
[0102] Y may comprise an antibody or antibody fragment that binds
to at least a portion of a receptor on a cell. Y may comprise an
antibody or antibody fragment that binds to at least a portion of a
co-receptor on a cell. Y may comprise an antibody or antibody
fragment that binds to at least a portion of a co-receptor on a B
cell. Y may comprise an antibody or antibody fragment that binds to
at least a portion of an antigen or cell surface marker on a cell.
The cell may be a hematopoietic cell. The hematopoietic cell may be
a myeloid cell. The myeloid cell may be an erythrocyte,
thrombocyte, neutrophil, monocyte, macrophage, eosinophil,
basophil, or mast cell. The cell may be a lymphoid cell. The
hematopoietic cell may be a lymphoid cell. The lymphoid cell may be
a B-cell, T-cell, or NK-cell. The hematopoietic cell may be a
leukocyte. The hematopoietic cell may be a lymphocyte. The cell may
be a cancerous cell. The cell may be a tumor cell. The cell may be
a leukemic cell. The cell may be a non-tumor cell. The cell may be
a prostate cell. The cell may be a breast cell. The cell may be a
glial cell. The cell may be a liver cell, kidney cell, lung cell,
cardiac cell, muscle cell, nerve cell, neuron, brain cell,
epithelial cell, esophageal cell.
[0103] Y may comprise an antibody or antibody fragment that binds
to a receptor on a cell. The receptor may be a growth factor
receptor. The growth factor receptor may be an epidermal growth
factor receptor (EGFR), platelet derived growth factor receptor
(PDGFR) or fibroblast growth factor receptor (FGFR). The EGFR may
be EGFR1. The EGFR may be Her2. The EGFR may be Her3 or Her4. The
EGFR may have a mutation. The EGFR may be EGFRvIII. The receptor
may be an interleukin receptor (ILR). The interleukin receptor may
be selected from IL1R, an IL2R, an IL3R, an IL4R, an IL5R, an IL6R,
an IL7R, an IL8R, an IL9R, an IL10R, an IL11R, an IL12R, an IL13R,
an IL14R, an IL15R and an IL17R. The interleukin receptor may be
IL13R.alpha.2.
[0104] Y may comprise an antibody or antibody fragment that binds
to an antigen or cell surface marker on a hematopoietic cell. Y may
comprise an antibody or antibody fragment that binds to an antigen
or cell surface marker on a B-cell. The antigen or cell surface
marker on the B-cell may be a B-lymphocyte antigen CD20 (CD20).
[0105] Y may comprise an antibody or antibody fragment that binds
to a differentiation antigen. The differentiation antigen may be a
CD44. The CD44 may be CD44v6. The differentiation antigen may be
CD33. CD33 may be highly expressed on cells of an acute myeloid
leukemia. CD33 may be expressed on a cancer stem cell.
[0106] Y may comprise an antibody or antibody fragment that binds
to a cell surface protein. The cell surface protein may be a cell
adhesion molecule. The cell adhesion molecule may be a cell surface
glycoprotein. The cell surface glycoprotein may be CS1, also
referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24. CS1
may co-localize with CD138. The cell adhesion molecule may be
PVRL4, also referred to as Nectin 4.
[0107] Y may comprise an antibody or antibody fragment that binds
to C-type lectin-like molecule-1. Y may comprise an antibody or
antibody fragment that binds a receptor tyrosine kinase-like orphan
receptor (ROR). The ROR may be ROR1. Y may comprise a bispecific
single chain monoclonal antibody. The bispecific single chain
monoclonal antibody may be bscWue1.
[0108] Y may comprise an antibody or at least a portion of an
antibody that is a human, fully human, humanized, human engineered,
non-human, or chimeric antibody. Y may comprise an antibody or at
least a portion of an antibody that is a mammalian antibody. Y may
comprise an antibody or at least a portion of an antibody that is a
non-mammalian antibody.
[0109] Y may comprise a sequence based on or derived from one or
more antibodies and/or antibody fragments sequences. Y may comprise
a sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more homologous to a
sequence based on or derived from one or more antibodies and/or
antibody fragments. Y may comprise a sequence that is at least
about 70% homologous to a sequence based on or derived from one or
more antibodies and/or antibody fragments. Y may comprise a
sequence that is at least about 80% homologous to a sequence based
on or derived from one or more antibodies and/or antibody
fragments. Y may comprise a sequence that is at least about 90%
homologous to a sequence based on or derived from one or more
antibodies and/or antibody fragments. Y may comprise a sequence
that is at least about 95% homologous to a sequence based on or
derived from one or more antibodies and/or antibody fragments. The
sequence may be a peptide sequence. Alternatively, the sequence is
a nucleotide sequence.
[0110] Y may comprise a peptide sequence that differs from a
peptide sequence based on or derived from one or more antibodies or
antibody fragments by less than or equal to about 20, 17, 15, 12,
10, 8, 6, 5, 4 or fewer amino acids. Y may comprise a peptide
sequence that differs from a peptide sequence based on or derived
from one or more antibodies or antibody fragments by less than or
equal to about 4 or fewer amino acids. Y may comprise a peptide
sequence that differs from a peptide sequence based on or derived
from one or more antibodies or antibody fragments by less than or
equal to about 3 or fewer amino acids. Y may comprise a peptide
sequence that differs from a peptide sequence based on or derived
from one or more antibodies or antibody fragments by less than or
equal to about 2 or fewer amino acids. Y may comprise a peptide
sequence that differs from a peptide sequence based on or derived
from one or more antibodies or antibody fragments by less than or
equal to about 1 or fewer amino acids. The amino acids may be
consecutive, nonconsecutive, or a combination thereof. For example,
Y may comprise a peptide sequence that differs from a peptide
sequence based on or derived from one or more antibodies or
antibody fragments by less than about 3 consecutive amino acids.
Alternatively, or additionally, Y may comprise a peptide sequence
that differs from a peptide sequence based on or derived from one
or more antibodies or antibody fragments by less than about 2
non-consecutive amino acids. In another example, Y may comprise a
peptide sequence that differs from a peptide sequence based on or
derived from one or more antibodies or antibody fragments by less
than about 5 amino acids, wherein 2 of the amino acids are
consecutive and 2 of the amino acids are non-consecutive.
[0111] Y may comprise a nucleotide sequence that differs from a
nucleotide sequence based on or derived from one or more antibodies
or antibody fragments by less than or equal to about 30, 25, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or fewer
nucleotides or base pairs. Y may comprise a nucleotide sequence
that differs from a nucleotide sequence based on or derived from
one or more antibodies or antibody fragments by less than or equal
to about 15 or fewer nucleotides or base pairs. Y may comprise a
nucleotide sequence that differs from a nucleotide sequence based
on or derived from one or more antibodies or antibody fragments by
less than or equal to about 12 or fewer nucleotides or base pairs.
Y may comprise a nucleotide sequence that differs from a nucleotide
sequence based on or derived from one or more antibodies or
antibody fragments by less than or equal to about 9 or fewer
nucleotides or base pairs. Y may comprise a nucleotide sequence
that differs from a nucleotide sequence based on or derived from
one or more antibodies or antibody fragments by less than or equal
to about 6 or fewer nucleotides or base pairs. Y may comprise a
nucleotide sequence that differs from a nucleotide sequence based
on or derived from one or more antibodies or antibody fragments by
less than or equal to about 4 or fewer nucleotides or base pairs. Y
may comprise a nucleotide sequence that differs from a nucleotide
sequence based on or derived from one or more antibodies or
antibody fragments by less than or equal to about 3 or fewer
nucleotides or base pairs. Y may comprise a nucleotide sequence
that differs from a nucleotide sequence based on or derived from
one or more antibodies or antibody fragments by less than or equal
to about 2 or fewer nucleotides or base pairs. Y may comprise a
nucleotide sequence that differs from a nucleotide sequence based
on or derived from one or more antibodies or antibody fragments by
less than or equal to about 1 or fewer nucleotides or base pairs.
The nucleotides or base pairs may be consecutive, nonconsecutive,
or a combination thereof. For example, Y may comprise a nucleotide
sequence that differs from a nucleotide sequence based on or
derived from one or more antibodies or antibody fragments by less
than about 3 consecutive nucleotides or base pairs. Alternatively,
or additionally, Y may comprise a nucleotide sequence that differs
from a nucleotide sequence based on or derived from one or more
antibodies or antibody fragments by less than about 2
non-consecutive nucleotides or base pairs. In another example, Y
may comprise a nucleotide sequence that differs from a nucleotide
sequence based on or derived from one or more antibodies or
antibody fragments by less than about 5 nucleotides or base pairs,
wherein 2 of the nucleotides or base pairs are consecutive and 2 of
the nucleotides or base pairs are non-consecutive.
[0112] The peptide sequence of Y may differ from the peptide
sequence of the antibody or antibody fragment that it is based on
and/or derived from by one or more amino acid substitutions. The
peptide sequence of Y may differ from the peptide sequence of the
antibody or antibody fragment that it is based on and/or derived
from by two or more amino acid substitutions. The peptide sequence
of Y may differ from the peptide sequence of the antibody or
antibody fragment that it is based on and/or derived from by three
or more amino acid substitutions. The peptide sequence of Y may
differ from the peptide sequence of the antibody or antibody
fragment that it is based on and/or derived from by four or more
amino acid substitutions. The peptide sequence of Y may differ from
the peptide sequence of the antibody or antibody fragment that it
is based on and/or derived from by five or more amino acid
substitutions. The peptide sequence of Y may differ from the
peptide sequence of the antibody or antibody fragment that it is
based on and/or derived from by six or more amino acid
substitutions. The peptide sequence of Y may differ from the
peptide sequence of the antibody or antibody fragment that it is
based on and/or derived from by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,
14, 15, 17, 20, 25 or more amino acid substitutions.
[0113] The nucleotide sequence of Y may differ from the nucleotide
sequence of the antibody or antibody fragment that it is based on
and/or derived from by one or more nucleotide and/or base pair
substitutions. The nucleotide sequence of Y may differ from the
nucleotide sequence of the antibody or antibody fragment that it is
based on and/or derived from by two or more nucleotide and/or base
pair substitutions. The nucleotide sequence of Y may differ from
the nucleotide sequence of the antibody or antibody fragment that
it is based on and/or derived from by three or more nucleotide
and/or base pair substitutions. The nucleotide sequence of Y may
differ from the nucleotide sequence of the antibody or antibody
fragment that it is based on and/or derived from by four or more
nucleotide and/or base pair substitutions. The nucleotide sequence
of Y may differ from the nucleotide sequence of the antibody or
antibody fragment that it is based on and/or derived from by five
or more nucleotide and/or base pair substitutions. The nucleotide
sequence of Y may differ from the nucleotide sequence of the
antibody or antibody fragment that it is based on and/or derived
from by six or more nucleotide and/or base pair substitutions. The
nucleotide sequence of Y may differ from the nucleotide sequence of
the antibody or antibody fragment that it is based on and/or
derived from by nine or more nucleotide and/or base pair
substitutions. The nucleotide sequence of Y may differ from the
nucleotide sequence of the antibody or antibody fragment that it is
based on and/or derived from by twelve or more nucleotide and/or
base pair substitutions. The nucleotide sequence of Y may differ
from the nucleotide sequence of the antibody or antibody fragment
that it is based on and/or derived from by fifteen or more
nucleotide and/or base pair substitutions. The nucleotide sequence
of Y may differ from the nucleotide sequence of the antibody or
antibody fragment that it is based on and/or derived from by
eighteen or more nucleotide and/or base pair substitutions. The
nucleotide sequence of Y may differ from the nucleotide sequence of
the antibody or antibody fragment that it is based on and/or
derived from by 20, 22, 24, 25, 27, 30 or more nucleotide and/or
base pair substitutions.
[0114] Y may comprise one or more unnatural amino acids. Y may
comprise two or more unnatural amino acids. Y may comprise three or
more unnatural amino acids. Y may comprise four or more unnatural
amino acids. Y may comprise 5, 6, 7, 8, 9, 10 or more unnatural
amino acids.
[0115] Y may be coupled to one or more linkers. Y may be linked to
X by one or more linkers. Y may be linked to X by two or more
linkers. Y may be linked to X by three or more linkers.
[0116] The distance between X and Y may be between about 1
angstroms (.ANG.) to about 120 angstroms (.ANG.). The distance
between X and Y may be between about 5 angstroms (.ANG.) to about
105 angstroms (.ANG.). The distance between X and Y may be between
about 10 angstroms (.ANG.) to about 100 angstroms (.ANG.). The
distance between X and Y may be between about 10 angstroms (.ANG.)
to about 90 angstroms (.ANG.). The distance between X and Y may be
between about 10 angstroms (.ANG.) to about 80 angstroms (.ANG.).
The distance between X and Y may be between about 10 angstroms
(.ANG.) to about 70 angstroms (.ANG.). The distance between X and Y
may be between about 15 angstroms (.ANG.) to about 45 angstroms
(.ANG.). The distance between X and Y may be equal to or greater
than about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 27, 30 or more angstroms. The distance between X and Y
may be equal to or greater than about 10 angstroms. The distance
between X and Y may be equal to or greater than about 15 angstroms.
The distance between X and Y may be equal to or greater than about
20 angstroms. The distance between X and Y may be equal to or less
than about 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43,
42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30 or fewer
angstroms. The distance between X and Y may be equal to or less
than about 100 angstroms. The distance between X and Y may be equal
to or less than about 80 angstroms. The distance between X and Y
may be equal to or less than about 60 angstroms. The distance
between X and Y may be equal to or less than about 40
angstroms.
II. Linkers
[0117] The bispecific antibodies disclosed herein may comprise one
or more linkers (e.g., L1, L2). Unless otherwise specified, a
linker may refer to a linker before it is coupled, reacted or
linked to an antibody, antibody fragment, or another linker (e.g.
L1", L2"), a linker that is coupled, reacted or linked to an
antibody or antibody fragment to produce an intermediate (e.g.
X-L1', L1'-X, Y-L2', L2'-Y) or a linker in a bispecific antibody
(e.g. X-L1-Y, X-L1-L2-Y, Y-L2-L1-X). The antibodies disclosed
herein may comprise two or more linkers. The antibodies disclosed
herein may comprise three or more linkers. The antibodies disclosed
herein may comprise 4, 5, 6, 7 or more linkers. The linker may
comprise one or more termini. The linker may comprise two termini.
The one or more termini may have a similar orthogonal reactivity.
The one or more termini may have a different orthogonal
reactivity.
[0118] The one or more linkers may comprise one or more reactive
functional groups that may react with a complementary reactive
functional group on another antibody or linker. The linker may be a
bifunctional linker. The bifunctional may comprise
BCN-TEG-ONH.sub.2, as depicted in FIG. 18. The bifunctional linker
may comprise N.sub.3-TEG-ONH.sub.2, as depicted in FIG. 18. The
bifunctional linker may be heterobifunctional, as exemplified in
FIG. 17B. The heterobifunctional linker may be Tet-TEG-ONH.sub.2 or
a derivation thereof. The heterobifunctional linker may be
TCO-TEC-ONH.sub.2 or a derivation thereof. The linker may comprise
ethylene glycol. The linker may be a bifunctional ethylene glycol
linker.
[0119] One or more linkers may be formed by reaction of an amino
acid on X with a linker already attached to Y. One or more linkers
may be formed by reaction of an amino acid on Y with a linker
already attached to X. One or more linkers may be formed by
reaction of a linker already attached to X with another linker
already attached to Y. In order to form a linker already attached
to X or Y, a bifunctional linker, with two orthogonally reactive
functional groups, may be coupled to X or Y, such that one
remaining reactive functional group is available for subsequent
coupling.
[0120] The linker may be the product of a bioorthogonal reaction.
The linker may comprise an oxime, a tetrazole, a Diels Alder
adduct, a hetero Diels Alder adduct, an aromatic substitution
reaction product, a nucleophilic substitution reaction product, an
ester, an amide, a carbamate, an ether, a thioether, or a Michael
reaction product. The linker may be a cycloaddition product, a
metathesis reaction product, a metal-mediated cross-coupling
reaction product, a radical polymerization product, an oxidative
coupling product, an acyl-transfer reaction product, or a photo
click reaction product. The cycloaddition may be a
Huisgen-cycloaddition. The cycloaddition may be a copper-free [3+2]
Huisgen-cycloaddition. The cycloaddition may be a Diels-Alder
reaction. The cycloaddition may be a hetero Diels-Alder reaction.
The linker may be the product of an enzyme-mediated reaction. The
linker may be a product of a transglutaminase-mediated reaction.
The linker may comprise a disulfide bridge that connects two
cysteine residues, such as ThioBridge.TM. technology by
PolyTherics. The linker may comprise a maleimide bridge that
connects two amino acid residues. The linker may comprise a
maleimide bridge that connects two cysteine residues.
[0121] Each of the one or more linkers may comprise one or more
ethylene glycols. Each of the one or more linkers may comprise at
least one reactive functional group selected from alkoxy-amine,
hydrazine, aryl/alkyl azide, alkyne, alkene, tetrazine,
dichlorotriazine, tresylate, succinimidyl carbonate, benzotriazole
carbonate, nitrophenyl carbonate, trichlorophenyl carbonate,
carbonylimidazole, succinimidyl succinate, maleimide, vinylsulfone,
haloacetamide, and disulfide. The alkene may be selected from
norbornene, trans-cyclooctene, and cyclopropene. Each of the one or
more linkers may comprise at least one alkoxy amine. Each of the
one or more linkers may comprise at least one azide. Each of the
one or more linkers may comprise at least one cyclooctyne. Each of
the one or more linkers may comprise at least one tetrazine.
[0122] The linker may couple with one or more natural amino acids
on X or Y. The linker may couple with one or more unnatural amino
acids on X or Y. The linker may couple with an amino acid which is
the product of site-specific mutagenesis. The linker may couple
with a cysteine which is the product of site-specific mutagenesis.
The linker (e.g., substituted maleimide) may couple with a cysteine
which is the product of site-specific mutagenesis, as well as a
native cysteine residue. Two linkers, each with complementary
reactive functional groups, may couple with one another.
[0123] The one or more linkers may comprise a cleavable linker. The
one or more linkers may comprise a non-cleavable linker. The one or
more linkers may comprise a flexible linker. The one or more
linkers may comprise an inflexible linker.
[0124] The one or more linkers may comprise an alkoxy-amine, azide
group and/or cyclooctyne group at one or more termini. The one or
more linkers may comprise an alkoxy-amine at one terminus and an
azide group at the other terminus. The one or more linkers may
comprise an alkoxy-amine at one terminus and a cyclooctyne group at
the other terminus.
[0125] The one or more linkers may be coupled to X, Y, or a
combination thereof. The one or more linkers may be coupled to X
and/or Y to form one or more intermediates of the Formula III:
L1'-X, Formula IIIA: X-L1', Formula IV: L2'-Y or Formula IVA:
Y-L2'. The one or more linkers may be coupled to X and/or Y to form
X-L1', Y-L2', L1'-X or L2'-Y. X-L1' may be coupled to Y. X-L1' may
be coupled to L2'-Y. X-L1' may be coupled to Y. X may be coupled to
L2'-Y. Y-L2' may be coupled to L1'-X. Y-L2' may be coupled to X. Y
may be coupled to X-L1'. The one or more linkers may be coupled to
X and/or Y by oxime formation. The one or more linkers may be
coupled to X and/or Y by a cyclooctyne, cyclopropene, arly/alkyl
azides, trans-cyclooctene, norborene, tetrazine bond, or a
combination thereof. The one or more linkers may be coupled to X
and/or Y by a covalent bond, non-covalent bond, ionic bond, or a
combination thereof.
[0126] The two or more linkers may be linked. The two or more
linkers may be linked through one or more copper-free reactions.
The two or more linkers may be linked through one or more
cycloadditions. The two or more linkers may be linked through one
or more Huisgen-cycloadditions. The two or more linkers may be
linked through one or more copper-free [3+2]
Huisgen-cycloadditions. The two or more linkers may be linked
through one or more copper-containing reactions. The two or more
linkers may be linked through one or more Diels Alder reactions.
The two or more linkers may be linked through one or more hetero
Diels Alder reactions.
[0127] The linker may possess a length that is sufficiently long to
allow the anti-CD3 antibody or antibody fragment and the second
antibody or antibody fragment to be linked without steric hindrance
from one another and sufficiently short to retain the intended
activity of the antibody. The intended activity of the antibody may
be to bring an effector cell and a target cell within a proximity
sufficient to allow the targeting cell to have an effect on the
target cell. The one or more linkers may be sufficiently
hydrophilic that it does not cause instability of the antibody. The
one or more linkers may be sufficiently hydrophilic that it does
not cause the antibody to be insoluble. The one or more linkers may
be sufficiently stable. The one or more linkers may be sufficiently
stable in vivo (e.g. it is not cleaved by serum, enzymes, etc.).
The one or more linkers may be between about 1 angstroms (.ANG.) to
about 120 angstroms (.ANG.) in length. The one or more linkers may
be between about 5 angstroms (.ANG.) to about 105 angstroms (.ANG.)
in length. The one or more linkers may be between about 10
angstroms (.ANG.) to about 100 angstroms (.ANG.) in length. The one
or more linkers may be between about 10 angstroms (.ANG.) to about
90 angstroms (.ANG.) in length. The one or more linkers may be
between about 10 angstroms (.ANG.) to about 80 angstroms (.ANG.) in
length. The one or more linkers may be between about 10 angstroms
(.ANG.) to about 70 angstroms (.ANG.) in length. The one or more
linkers may be between about 15 angstroms (.ANG.) to about 45
angstroms (.ANG.) in length. The one or more linkers may be equal
to or greater than about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 27, 30 or more angstroms in length. The one
or more linkers may be equal to or greater than about 10 angstroms
in length. The one or more linkers may be equal to or greater than
about 15 angstroms in length. The one or more linkers may be equal
to or greater than about 20 angstroms in length. The one or more
linkers may be equal to or less than about 110, 100, 90, 85, 80,
75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34,
33, 32, 31, 30 or fewer angstroms in length. The one or more
linkers may be equal to or less than about 100 angstroms in length.
The one or more linkers may be equal to or less than about 80
angstroms in length. The one or more linkers may be equal to or
less than about 60 angstroms in length. The one or more linkers may
be equal to or less than about 40 angstroms in length.
[0128] The total length of the linkers may be between about 1
angstroms (.ANG.) to about 120 angstroms (.ANG.). The total length
of the linkers may be between about 5 angstroms (.ANG.) to about
105 angstroms (.ANG.). The total length of the linkers may be
between about 10 angstroms (.ANG.) to about 100 angstroms (.ANG.).
The total length of the linkers may be between about 10 angstroms
(.ANG.) to about 90 angstroms (.ANG.). The total length of the
linkers may be between about 10 angstroms (.ANG.) to about 80
angstroms (.ANG.). The total length of the linkers may be between
about 10 angstroms (.ANG.) to about 70 angstroms (.ANG.). The total
length of the linkers may be between about 15 angstroms (.ANG.) to
about 45 angstroms (.ANG.). The total length of the linkers may be
equal to or greater than about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or more angstroms. The total
length of the linkers may be equal to or greater than about 10
angstroms. The total length of the linkers may be equal to or
greater than about 15 angstroms. The total length of the linkers
may be equal to or greater than about 20 angstroms. The total
length of the linkers may be equal to or less than about 110, 100,
90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37,
36, 35, 34, 33, 32, 31, 30 or fewer angstroms. The total length of
the linkers may be equal to or less than about 100 angstroms. The
total length of the linkers may be equal to or less than about 80
angstroms. The total length of the linkers may be equal to or less
than about 60 angstroms. The total length of the linkers may be
equal to or less than about 40 angstroms.
III. Unnatural Amino Acids
[0129] The bispecific antibodies disclosed herein may comprise one
or more unnatural amino acids. As used herein, the terms "unnatural
amino acid" and "non-natural amino acid" may be used
interchangeably and may refer to non-proteinogenic amino acids that
either occur naturally or are chemically synthesized. The
bispecific antibodies of Formulas I, IA, II, and/or HA disclosed
herein may comprise X, wherein X comprises one or more unnatural
amino acids. The bispecific antibodies of Formulas I, IA, II,
and/or IIA disclosed herein may comprise X, wherein X does not
comprise an unnatural amino acid. The bispecific antibodies of
Formulas I, IA, II, and/or IIA disclosed herein may comprise Y,
wherein Y comprises one or more unnatural amino acids. The
bispecific antibodies of Formulas I, IA, II, and/or HA disclosed
herein may comprise Y, wherein Y does not comprise an unnatural
amino acid. The bispecific antibodies of Formulas I, IA, II, and/or
HA disclosed herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more unnatural amino acids. The antibodies disclosed herein may
comprise at least one antibody or antibody fragment that comprises
one or more unnatural amino acids. The antibodies disclosed herein
may comprise at least two antibody or antibody fragments that
independently comprise at least one unnatural amino acid.
[0130] The one or more unnatural amino acids may be incorporated
into a bispecific antibody (e.g., antibody of Formula I, IA, II,
and IIA); intermediate (e.g., intermediate of Formula III, IIIA,
IV, IVA); X antibody or antibody fragment; Y antibody or antibody
fragment; or a combination thereof. The one or more unnatural amino
acids may be site-specifically incorporated into an antibody (e.g.,
antibody of Formula I, IA, II, and IIA); intermediate (e.g.,
intermediate of Formula III, IIIA, IV, IVA); X antibody or antibody
fragment; Y antibody or antibody fragment; or a combination
thereof. The one or more unnatural amino acids may be incorporated
into an antibody fragment of an antibody (e.g., antibody of Formula
I, IA, II, and HA); intermediate (e.g., intermediate of Formula
III, IIIA, IV, IVA); X antibody or antibody fragment; Y antibody or
antibody fragment; or a combination thereof. The one or more
unnatural amino acids may be site-specifically incorporated into an
antibody fragment of an antibody (e.g., antibody of Formula I, IA,
II, and HA); intermediate (e.g., intermediate of Formula III, IIIA,
IV, IVA); X antibody or antibody fragment; Y antibody or antibody
fragment; or a combination thereof.
[0131] The one or more unnatural amino acids may be incorporated
into a heavy chain, light chain, variable region, constant region,
Fab fragment of an antibody (e.g., antibody of Formula I, IA, II,
and HA); intermediate (e.g., intermediate of Formula III, IIIA, IV,
IVA); X antibody or antibody fragment; Y antibody or antibody
fragment; or a combination thereof.
[0132] The one or more unnatural amino acids may be encoded by a
codon that does not code for one of the twenty natural amino acids.
The one or more unnatural amino acids may be encoded by a nonsense
codon (stop codon). The stop codon may be an amber codon. The amber
codon may comprise a UAG sequence. The stop codon may be an ochre
codon. The ochre codon may comprise a UAA sequence. The stop codon
may be an opal or umber codon. The opal or umber codon may comprise
a UGA sequence. The one or more unnatural amino acids may be
encoded by a four-base codon.
[0133] The one or more unnatural amino acids may be
p-acetylphenylalanine (pAcF or pAcPhe). The one or more unnatural
amino cids may be p-fluorophenylalanine (pFPhe). The one or more
unnatural amino acids may be selected from the group comprising
p-azidophenylalanine (pAzF), p-benzoylphenylalanine (pBpF),
p-propargyloxyphenylalanine (pPrF), p-iodophenylalanine (pIF),
p-cyanophenylalanine (pCNF), p-carboxylmethylphenylalanine (pCmF),
3-(2-naphthyl)alanine (NapA), p-boronophenylalanine (pBoF),
o-nitrophenylalanine (oNiF), (8-hydroxyquinolin-3-yl)alanine (HQA),
selenocysteine, and (2,2'-bipyridin-5-yl)alanine (BipyA).
[0134] The one or more unnatural amino acids may be .beta.-amino
acids (.beta.3 and (.beta.2), homo-amino acids, proline and pyruvic
acid derivatives, 3-substituted alanine derivatives, glycine
derivatives, ring-substituted phenylalanine and tyrosine
derivatives, linear core amino acids, diamino acids, D-amino acids,
N-methyl amino acids, or a combination thereof.
[0135] Additional examples of unnatural amino acids include, but
are not limited to, 1) various substituted tyrosine and
phenylalanine analogues such as O-methyl-L-tyrosine,
p-amino-L-phenylalanine, 3-nitro-L-tyrosine,
p-nitro-L-phenylalanine, m-methoxy-L-phenylalanine and
p-isopropyl-L-phenylalanine; 2) amino acids with aryl azide and
benzophenone groups that may be photo-cross-linked; 3) amino acids
that have unique chemical reactivity including
acetyl-L-phenylalanine and m-acetyl-L-phenylalanine,
O-allyl-L-tyrosine, O-(2-propynyl)-L-tyrosine,
p-ethylthiocarbonyl-L-phenylalanine and
p-(3-oxobutanoyl)-L-phenylalanine; 4) heavy-atom-containing amino
acids for phasing in X-ray crystallography including p-iodo and
p-bromo-L-phenylalanine; 5) the redox-active amino acid
dihydroxy-L-phenylalanine; 6) glycosylated amino acids including
b-N-acetylglucosamine-O-serine and
a-N-acetylgalactosamine-O-threonine; 7) fluorescent amino acids
with naphthyl, dansyl, and 7-aminocoumarin side chains; 8)
photocleavable and photoisomerizable amino acids with azobenzene
and nitrobenzyl Cys, Ser, and Tyr side chains; 9) the
phosphotyrosine mimetic p-carboxymethyl-L-phenylalanine; 10) the
glutamine homologue homoglutamine; and 11) 2-aminooctanoic
acid.
[0136] Additional unnatural amino acids are disclosed in Liu et al.
(Annu Rev Biochem, 79:413-44, 2010), Wang et al. (Angew Chem Int
Ed, 44:34-66, 2005) and PCT application numbers PCT/US2012/039472,
PCT/US2012/039468, PCT/US2007/088009, PCT/US2009/058668,
PCT/US2007/089142, PCT/US2007/088011, PCT/US2007/001485,
PCT/US2006/049397, PCT/US2006/047822 and PCT/US2006/044682, all of
which are incorporated by reference in their entireties.
[0137] The one or more unnatural amino acids may comprise at least
one oxime, carbonyl, dicarbonyl, hydroxylamine group or a
combination thereof. The one or more unnatural amino acids may
comprise at least one carbonyl, dicarbonyl, alkoxy-amine,
hydrazine, acyclic alkene, acyclic alkyne, cyclooctyne, aryl/alkyl
azide, norbornene, cyclopropene, trans-cyclooctene, or tetrazine
functional group or a combination thereof. The one or more
unnatural amino acids may be incorporated into X and/or Y by
methods known in the art. Cell-based or cell-free systems may be
used to alter the genetic sequence of X and/or Y, thereby producing
X and/or Y with one or more unnatural amino acids. Auxotrophic
strains may be used in place of engineered tRNA and synthetase. The
one or more unnatural amino acids may be produced through selective
reaction of one or more natural amino acids. The selective reaction
may be mediated by one or more enzymes. In one non-limiting
example, the selective reaction of one or more cysteines with
formylglycine generating enzyme (FGE) may produce one or more
formylglycines.
[0138] The one or more unnatural amino acids may take part in a
chemical reaction to form a linker. The chemical reaction to form
the linker may be a bioorthogonal reaction. The chemical reaction
to form the linker may be click chemistry.
IV. Antibody Compositions
[0139] Disclosed herein are compositions comprising one or more
bispecific antibodies disclosed herein. The compositions may
comprise a bispecific antibody comprising an anti-CD3 antibody or
anti-CD3 antibody fragment and a second antibody or antibody
fragment, wherein the anti-CD3 antibody or anti-CD3 antibody
fragment is site-specifically connected to the second antibody or
antibody fragment. The compositions may comprise a bispecific
antibody comprising (a) an anti-CD3 antibody or anti-CD3 antibody
fragment; (b) a second antibody or antibody fragment; and (c) one
or more linkers, wherein the one or more linkers links the anti-CD3
antibody or anti-CD3 antibody fragment to the second antibody or
antibody fragment, wherein the anti-CD3 antibody or anti-CD3
antibody fragment is site-specifically linked to the second
antibody or antibody fragment. The anti-CD3 antibody or anti-CD3
antibody fragment may comprise one or more unnatural amino acids.
The second antibody or antibody fragment may comprise one or more
unnatural amino acids. The anti-CD3 antibody or anti-CD3 antibody
fragment and the second antibody or antibody fragment may comprise
one or more unnatural amino acids. The composition may further
comprise one or more pharmaceutically acceptable excipients. The
composition may further comprise one or more solvents or
diluents.
[0140] The composition may comprise a bispecific antibody of
Formula I: X-L1-Y, wherein (i) X comprises an anti-CD3 antibody or
anti-CD3 antibody fragment; (ii) L1 comprises a linker; and (iii) Y
comprises a second antibody or antibody fragment. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise one or more
unnatural amino acids. The second antibody or antibody fragment may
comprise one or more unnatural amino acids. The anti-CD3 antibody
or anti-CD3 antibody fragment and the second antibody or antibody
fragment may comprise one or more unnatural amino acids. The
composition may further comprise one or more pharmaceutically
acceptable excipients. The composition may further comprise one or
more solvents or diluents.
[0141] The composition may comprise a bispecific antibody of
Formula IA: Y-L1-X, wherein (i) X comprises an anti-CD3 antibody or
anti-CD3 antibody fragment; (ii) L1 comprises a linker; and (iii) Y
comprises a second antibody or antibody fragment. The anti-CD3
antibody or anti-CD3 antibody fragment may comprise one or more
unnatural amino acids. The second antibody or antibody fragment may
comprise one or more unnatural amino acids. The anti-CD3 antibody
or anti-CD3 antibody fragment and the second antibody or antibody
fragment may comprise one or more unnatural amino acids. The
composition may further comprise one or more pharmaceutically
acceptable excipients. The composition may further comprise one or
more solvents or diluents.
[0142] The composition may comprise a bispecific antibody of
Formula II: X-L1-L2-Y, wherein (i) X comprises an anti-CD3 antibody
or anti-CD3 antibody fragment; (ii) L1 and L2 comprise a linker;
and (iii) Y comprises a second antibody or antibody fragment. The
anti-CD3 antibody or anti-CD3 antibody fragment may comprise one or
more unnatural amino acids. The second antibody or antibody
fragment may comprise one or more unnatural amino acids. The
anti-CD3 antibody or anti-CD3 antibody fragment and the second
antibody or antibody fragment may comprise one or more unnatural
amino acids. The composition may further comprise one or more
pharmaceutically acceptable excipients. The composition may further
comprise one or more solvents or diluents.
[0143] The composition may comprise a bispecific antibody of
Formula IIA: Y-L2-L1-X, wherein (i) X comprises an anti-CD3
antibody or anti-CD3 antibody fragment; (ii) L1 and L2 comprise a
linker; and (iii) Y comprises a second antibody or antibody
fragment. The anti-CD3 antibody or anti-CD3 antibody fragment may
comprise one or more unnatural amino acids. The second antibody or
antibody fragment may comprise one or more unnatural amino acids.
The anti-CD3 antibody or anti-CD3 antibody fragment and the second
antibody or antibody fragment may comprise one or more unnatural
amino acids. The composition may further comprise one or more
pharmaceutically acceptable excipients. The composition may further
comprise one or more solvents or diluents.
[0144] The term "pharmaceutically acceptable" as used herein,
refers to a material that does not abrogate the biological activity
or properties of the agents described herein, and is relatively
nontoxic (i.e., the toxicity of the material significantly
outweighs the benefit of the material). In some instances, a
pharmaceutically acceptable material may be administered to an
individual without causing significant undesirable biological
effects or significantly interacting in a deleterious manner with
any of the components of the composition in which it is
contained.
[0145] Pharmaceutical compositions herein may be formulated using
one or more physiologically acceptable carriers including
excipients and auxiliaries which facilitate processing of the
active agents into preparations which are used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen. A summary of pharmaceutical compositions is found, for
example, in Remington: The Science and Practice of Pharmacy,
Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover,
John E., Remington's Pharmaceutical Sciences, Mack Publishing Co.,
Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds.,
Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980;
and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh
Ed. (Lippincott Williams & Wilkins, 1999).
[0146] A pharmaceutical composition disclosed herein may further
comprise a pharmaceutically acceptable diluent(s), excipient(s), or
carrier(s). The pharmaceutical compositions may include other
medicinal or pharmaceutical agents, carriers, adjuvants, such as
preserving, stabilizing, wetting or emulsifying agents, solution
promoters, salts for regulating the osmotic pressure, and/or
buffers. In addition, the pharmaceutical compositions also contain
other therapeutically valuable substances.
[0147] A pharmaceutical composition disclosed herein may be
administered to a subject by any suitable administration route,
including but not limited to, parenteral (intravenous,
subcutaneous, intraperitoneal, intramuscular, intravascular,
intrathecal, intravitreal, infusion, or local), topical, oral, or
nasal administration. A pharmaceutical composition disclosed herein
may be administered to a subject by an intranasal administration. A
pharmaceutical composition disclosed herein may be administered to
a subject by a microneedle device. The microneedle device may be
used to deliver a low dosage of the pharmaceutical composition due
to the relatively high potency or efficacy of the antibody. The
microneedle device may be a microneedle device as described in U.S.
Pat. No. 7,416,541, Peters et al., Pharm Res 29:1618-26 (2012), and
Daddona et al., Pharm Res. 28:159-65 (2011).
[0148] Formulations suitable for intramuscular, subcutaneous,
peritumoral, or intravenous injection may include physiologically
acceptable sterile aqueous or non-aqueous solutions, dispersions,
suspensions or emulsions, and sterile powders for reconstitution
into sterile injectable solutions or dispersions. Examples of
suitable aqueous and non-aqueous carriers, diluents, solvents, or
vehicles including water, ethanol, polyols (propyleneglycol,
polyethylene-glycol, glycerol, cremophor and the like), suitable
mixtures thereof, vegetable oils (such as olive oil) and injectable
organic esters such as ethyl oleate. Proper fluidity is maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersions, and by the use of surfactants. Formulations suitable
for subcutaneous injection also contain optional additives such as
preserving, wetting, emulsifying, and dispensing agents.
[0149] For intravenous injections, an active agent may be
optionally formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological saline buffer.
[0150] Parenteral injections optionally involve bolus injection or
continuous infusion. Formulations for injection are optionally
presented in unit dosage form, e.g., in ampoules or in multi dose
containers, with an added preservative. The pharmaceutical
composition described herein may be in a form suitable for
parenteral injection as a sterile suspensions, solutions or
emulsions in oily or aqueous vehicles, and contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include
aqueous solutions of an active agent in water soluble form.
Additionally, suspensions are optionally prepared as appropriate
oily injection suspensions.
[0151] The pharmaceutical composition described herein may be in
unit dosage forms suitable for single administration of precise
dosages. In unit dosage form, the formulation may divided into unit
doses containing appropriate quantities of an active agent
disclosed herein. The unit dosage may be in the form of a package
containing discrete quantities of the formulation. Non-limiting
examples are packaged tablets or capsules, and powders in vials or
ampoules. Aqueous suspension compositions may be packaged in
single-dose non-reclosable containers. Alternatively, multiple-dose
reclosable containers are used, in which case it is typical to
include a preservative in the composition. By way of example only,
formulations for parenteral injection are presented in unit dosage
form, which include, but are not limited to ampoules, or in multi
dose containers, with an added preservative.
V. Antibody Production Methods
[0152] Disclosed herein are methods of producing bispecific
antibodies comprising connecting an anti-CD3 antibody or anti-CD3
antibody fragment and a second antibody or antibody fragment
site-specifically. Disclosed herein are methods of producing the
antibody of Formula I: X-L1-Y or Formula IA: Y-L1-X, comprising
coupling L1'' to X to produce an intermediate of Formula III: X-L1'
or Formula IIIA: L1'-X, wherein X comprises an anti-CD3 antibody or
anti-CD3 antibody fragment; and coupling the intermediate to Y;
wherein Y comprises at least a portion of a second antibody or
antibody fragment; thereby producing the antibody of Formula I or
IA.
[0153] Disclosed herein are methods of producing bispecific
antibodies of Formula I: X-L1-Y or Formula IA: Y-L1-X, comprising
coupling L1'' to Y to produce an intermediate of Formula IV: Y-L1'
or Formula IVA: L1'-Y, wherein Y comprises at least a portion of a
second antibody or antibody fragment; and coupling the intermediate
to X, wherein X comprises an anti-CD3 antibody or anti-CD3 antibody
fragment, thereby producing the antibody of Formula I or IA. The
method may further comprise incorporating one or more unnatural
amino acids into X and/or Y. The method may further comprise
incorporating one or more unnatural amino acids into X and/or Y
site-specifically.
[0154] Disclosed herein are methods of producing bispecific
antibodies of Formula II: X-L1-L2-Y or Formula IIA: Y-L1-L2-X,
comprising: (a) coupling L1'' to X to produce a first intermediate
of Formula III: X-L1' or Formula IIIA: L1'-X, wherein X comprises
an anti-CD3 antibody or anti-CD3 antibody fragment; (b) coupling
L2'' to Y to produce a second intermediate of Formula IV: L2'-Y or
Formula IVA: Y-L2', wherein Y comprises at least a portion of a
second antibody or antibody fragment; and (c) linking the first
intermediate to the second intermediate, thereby producing the
antibody of Formula II or IIA. The method may further comprise
incorporating one or more unnatural amino acids into X and/or Y.
The method may further comprise incorporating one or more unnatural
amino acids into X and/or Y site-specifically. Coupling L1'' to X
may comprise coupling L1'' to X site-specifically. Coupling L2'' to
Y may comprise coupling L2'' to Y site-specifically. Coupling L1''
to X and coupling L2'' to Y may comprise coupling L1'' to X
site-specifically and coupling L2'' to Y site-specifically. The
method may further comprise coupling L1''/L2'' to Y at the one or
more unnatural amino acids of Y. The method may further comprise
coupling L1'' to X at the one or more unnatural amino acids of
Y.
[0155] Disclosed herein are methods of coupling one or more first
linkers to an anti-CD3 antibody or anti-CD3 antibody fragment to
produce a first intermediate; coupling one or more second linkers
to a second antibody or antibody fragment; and coupling the first
intermediate to the second intermediate via the linkers. The
anti-CD3 antibody or anti-CD3 antibody fragment may comprise an
unnatural amino acid. The second antibody or antibody fragment may
comprise an unnatural amino acid. Coupling one or more first
linkers to the anti-CD3 antibody or anti-CD3 antibody fragment to
produce the first intermediate may comprise forming a stable oxime
between the anti-CD3 antibody or anti-CD3 antibody fragment and the
linker. Coupling one or more second linkers to the second antibody
or antibody fragment to produce the second intermediate may
comprise forming a stable oxime between the second antibody or
antibody fragment and the linker. The oxime may be formed between
an alkoxy-amine group of the linker and the unnatural amino acid.
The oxime may be formed between an alkoxy-amine group of the linker
and a ketone of the unnatural amino acid. The oxime may be formed
between an alkoxy-amine group of the linker and a ketone of the
unnatural amino acid, wherein the unnatural amino acid is
p-acetylphenylalanine (pAcF). The oxime may be between an
alkoxy-amine group of the first linker and the unnatural amino acid
of the anti-CD3 antibody or anti-CD3 antibody fragment. The oxime
may be formed between an alkoxy-amine group of the second linker
and the unnatural amino acid of the second antibody. The
first/second linker may be a bifunctional linker. The bifunctional
linker may comprise an azide group. The bifunctional linker may
comprise a cyclooctyne group. The azide group may be linked to the
cyclooctyne group through a chemical reaction. The chemical
reaction may be a copper-free [3+2] Huisgen-cycloaddition ("Click"
reaction). The first/second linker may be a heterobifunctional
linker. The heterobifunctional linker may comprise two reactive
functional groups selected from alkoxy-amine, hydrazine, aryl/alkyl
azide, alkyne, alkene, tetrazine, dichlorotriazine, tresylate,
succinimidyl carbonate, benzotriazole carbonate, nitrophenyl
carbonate, trichlorophenyl carbonate, carbonylimidazole,
succinimidyl succinate, maleimide, vinylsulfone, haloacetamide, and
disulfide.
[0156] The anti-CD3 antibody may not comprise an unnatural amino
acid while the second antibody or antibody fragment does comprise
an unnatural amino acid. Coupling the one or more linkers to the
anti-CD3 antibody or anti-CD3 antibody fragment may comprise
forming a stable oxime between the anti-CD3 antibody or anti-CD3
antibody fragment and one or more of the first linkers to form the
first intermediate. The oxime may be formed between an alkoxy-amine
group of the first linker and the natural amino acid of the
anti-CD3 antibody or anti-CD3 antibody fragment. Coupling one or
more second linkers to the second antibody or antibody fragment to
produce the second intermediate may comprise forming a stable oxime
between the second antibody or antibody fragment and the one or
more second linkers. The oxime may be formed between an
alkoxy-amine group of the second linker and the unnatural amino
acid of the second antibody or antibody fragment. The first/second
linker may be a bifunctional linker. The bifunctional linker may
comprise an azide group. The bifunctional linker may comprise a
cyclooctyne group. The azide group may be linked to the cyclooctyne
group through a chemical reaction. The chemical reaction may be a
copper-free [3+2] Huisgen-cycloaddition ("Click" reaction). The
first/second linker may be a heterobifunctinoal linker. The
heterobifunctional linker may comprise two reactive functional
groups selected from alkoxy-amine, hydrazine, aryl/alkyl azide,
alkyne, alkene, tetrazine, dichlorotriazine, tresylate,
succinimidyl carbonate, benzotriazole carbonate, nitrophenyl
carbonate, trichlorophenyl carbonate, carbonylimidazole,
succinimidyl succinate, maleimide, vinylsulfone, haloacetamide, and
disulfide.
[0157] The anti-CD3 antibody or anti-CD3 antibody fragment may
comprise an unnatural amino acid, while the second antibody does
not comprise an unnatural amino acid. Coupling the one or more
linkers to second antibody or antibody fragment may comprise
forming a stable oxime between the second antibody or antibody
fragment and one or more of the second linkers to form the second
intermediate. The oxime may be formed between an alkoxy-amine group
of the second linker and the natural amino acid. Coupling one or
more first linkers to the anti-CD3 antibody or anti-CD3 antibody
fragment to produce the first intermediate may comprise forming a
stable oxime between the anti-CD3 antibody or antibody fragment and
the one or more first linkers. The oxime may be formed between an
alkoxy-amine group of the first linker and the unnatural amino
acid. The first/second linker may be a bifunctional linker. The
bifunctional linker may comprise an azide group. The bifunctional
linker may comprise a cyclooctyne group. The azide group may be
linked to the cyclooctyne group through a chemical reaction. The
chemical reaction may be a copper-free [3+2] Huisgen-cycloaddition
("Click" reaction). The first/second linker may be a
heterobifunctional linker. The heterobifunctional linker may
comprise two reactive functional groups selected from alkoxy-amine,
hydrazine, aryl/alkyl azide, alkyne, alkene, tetrazine,
dichlorotriazine, tresylate, succinimidyl carbonate, benzotriazole
carbonate, nitrophenyl carbonate, trichlorophenyl carbonate,
carbonylimidazole, succinimidyl succinate, maleimide, vinylsulfone,
haloacetamide, and disulfide.
[0158] Conjugating the anti-CD3 antibody or anti-CD3 antibody
fragment to the second antibody or antibody fragment may comprise
one or more Huisgen-cycloadditions. Conjugating the anti-CD3
antibody or antibody fragment to the second antibody or antibody
fragment may comprise one or more copper-free [3+2]
Huisgen-cycloadditions. Conjugating the anti-CD3 antibody or
anti-CD3 antibody fragment to the second antibody or antibody
fragment may comprise one or more copper-containing reactions.
Conjugating the anti-CD3 antibody or anti-CD3 antibody fragment to
the second antibody or antibody fragment may comprise one or more
Diels Alder reactions. Conjugating the anti-CD3 antibody or
antibody fragment to the second antibody or antibody fragment may
comprise one or more hetero Diels Alder reactions.
[0159] Conjugating the first intermediate to the second
intermediate may comprise one or more Huisgen-cycloadditions.
Conjugating the first intermediate to the second intermediate may
comprise one or more copper-free [3+2] Huisgen-cycloadditions.
Conjugating the first intermediate to the second intermediate may
comprise one or more copper-containing reactions. Conjugating the
first intermediate to the second intermediate may comprise one or
more Diels Alder reactions. Conjugating the first intermediate to
the second intermediate may comprise one or more hetero Diels Alder
reactions.
[0160] Conjugating the one or more of the first linkers to the one
or more of the second linkers may comprise one or more
Huisgen-cycloadditions. Conjugating the one or more of the first
linkers to the one or more of the second linkers may comprise one
or more copper-free [3+2] Huisgen-cycloadditions. Conjugating the
one or more of the first linkers to the one or more of the second
linkers may comprise one or more copper-containing reactions.
Conjugating the one or more of the first linkers to the one or more
of the second linkers may comprise one or more Diels Alder
reactions. Conjugating the one or more of the first linkers to the
one or more of the second linkers may comprise one or more hetero
Diels Alder reactions.
[0161] Further disclosed herein is a method of producing the
bispecific antibody of Formula II: X-L1-L2-Y, comprising (a)
coupling L1'' to X to produce a first intermediate of Formula IIIA:
X-L1', wherein X comprises at least a portion of an anti-CD3
antibody or anti-CD3 antibody fragment; (b) coupling L2'' to Y to
produce a second intermediate of Formula IV: L2'-Y, wherein Y
comprises at least a portion of an antibody or antibody fragment;
and (c) linking the first intermediate of Formula III to the second
intermediate of Formula IV, thereby producing the antibody of
Formula II. The method may further comprise incorporating one or
more unnatural amino acids into X and/or Y.
VA. Incorporation of Unnatural Amino Acids
[0162] Incorporating one or more unnatural amino acids into an
antibody or antibody fragment may comprise modifying one or more
amino acid residues in an antibody or antibody fragment. Modifying
the one or more amino acid residues in an antibody or antibody
fragment may comprise mutating one or more nucleotides in the
nucleotide sequence encoding the antibody or antibody fragment.
Mutating the one or more nucleotides in the nucleotide sequence
encoding the antibody or antibody fragment may comprise altering a
codon encoding an amino acid to a nonsense codon.
[0163] Incorporating one or more unnatural amino acids into an
antibody or antibody fragment may comprise modifying one or more
amino acid residues in an antibody or antibody fragment to produce
one or more amber codons in an antibody or antibody fragment.
[0164] The one or more unnatural amino acids may incorporated into
an antibody or antibody fragment in response to an amber codon. The
one or more unnatural amino acids may be site-specifically
incorporated into an antibody or antibody fragment.
[0165] Incorporating one or more unnatural amino acids into an
antibody or antibody fragment may comprise use of one or more
genetically encoded unnatural amino acids with orthogonal chemical
reactivity relative to the canonical twenty amino acids to
site-specifically modify antibody fragments. Incorporating the one
or more unnatural amino acids may comprise use of an evolved
tRNA/aminoacyl-tRNA synthetase pair to site-specifically
incorporate one or more unnatural amino acids at defined sites in
the antibody or antibody fragment in response to one or more amber
nonsense codon.
[0166] Additional methods for incorporating unnatural amino acids
include, but are not limited to, methods disclosed in Chatterjee et
al. (A Versatile Platform for Single- and Multiple-Unnatural Amino
Acid Mutagenesis in Escherichia coli, Biochemistry, 2013), Kazane
et al. (J Am Chem Soc, 135(1):340-6, 2013), Kim et al. (J Am Chem
Soc, 134(24):9918-21, 2012), Johnson et al. (Nat Chem Biol,
7(11):779-86, 2011) and Hutchins et al. (J Mol Biol,
406(4):595-603, 2011).
VB. Coupling of Linkers
[0167] The methods disclosed herein may comprise coupling one or
more linkers to one or more antibodies, antibody fragments, or
combinations thereof to produce one or more antibody-linker and/or
antibody fragment-linker molecules. The methods may comprise
coupling a first linker to a first antibody or antibody fragment to
produce an antibody-linker molecule or antibody fragment-linker
molecule. The methods may comprise coupling a second linker to a
second antibody or antibody fragment to produce an antibody-linker
molecule or antibody fragment-linker molecule.
[0168] Coupling of the one or more linkers to the antibody or
antibody fragment may occur simultaneously. Coupling of the one or
more linkers to the antibody or antibody fragment may occur
sequentially. Coupling of the one or more linkers to the antibody
or antibody fragment may occur in a single reaction volume.
Coupling of the one or more linkers to the antibody or antibody
fragment may occur in two or more reaction volumes.
[0169] Coupling one or more linkers to an antibody or antibody
fragment may comprise forming one or more oximes between the linker
and the antibody or antibody fragment. Coupling one or more linkers
to an antibody or antibody fragment may comprise forming one or
more stable bonds between linker and the antibody or antibody
fragment. Coupling one or more linkers to an antibody or antibody
fragment may comprise forming one or more covalent bonds between
linker and the antibody or antibody fragment. Coupling one or more
linkers to an antibody or antibody fragment may comprise forming
one or more non-covalent bonds between linker and the antibody or
antibody fragment. Coupling one or more linkers to an antibody or
antibody fragment may comprise forming one or more ionic bonds
between linker and the antibody or antibody fragment.
VC. Linking Antibodies and Antibody Fragments
[0170] The methods may comprise linking the antibody, antibody
fragment and/or intermediates thereof to produce a bispecific
antibody comprising (a) an anti-CD3 antibody or anti-CD3 antibody
fragment; (b) a second antibody or antibody fragment; and (c) one
or more linkers, wherein the one or more linkers link the anti-CD3
antibody or anti-CD3 antibody fragment to the second antibody or
antibody fragment. The anti-CD3 antibody or anti-CD3 antibody
fragment may comprise one or more unnatural amino acids Linking the
anti-CD3 antibody or anti-CD3 antibody fragment to the second
antibody or antibody fragment may comprise conducting one or more
copper-free reactions Linking the anti-CD3 antibody or anti-CD3
antibody fragment to the second antibody or antibody fragment may
comprise conducting one or more copper-containing reactions Linking
the anti-CD3 antibody or anti-CD3 antibody fragment to the second
antibody or antibody fragment may comprise one or more
cycloadditions. Linking the anti-CD3 antibody or anti-CD3 antibody
fragment to the second antibody or antibody fragment may comprise
one or more Huisgen-cycloadditions Linking the anti-CD3 antibody or
anti-CD3 antibody fragment to the second antibody or antibody
fragment may comprise one or more Diels Alder reactions. Linking
the anti-CD3 antibody or anti-CD3 antibody fragment to the second
antibody or antibody fragment may comprise one or more Hetero Diels
Alder reaction.
VD. Purification of Antibodies
[0171] The methods may further comprise purifying the bispecific
antibody comprising (a) an anti-CD3 antibody or antibody fragment
comprising one or more unnatural amino acids; (b) a second antibody
or antibody fragment; and (c) one or more linkers, wherein the one
or more linkers link the anti-CD3 antibody or antibody fragment to
the second antibody or antibody fragment. The methods may further
comprise purifying one or more intermediates of the antibody or
antibody fragment antibody or antibody fragment (e.g.,
antibody-linker or antibody fragment-linker). Purifying the
antibody or intermediates may comprise removal of excess linkers,
non-linked antibodies or non-linked antibody fragments. Purifying
the antibody or intermediates may comprise use of one or more
concentrator columns, electrophoresis, filtration, centrifugation,
chromatography or a combination thereof. Chromatography may
comprise size-exclusion chromatography. Additional chromatography
methods include, but are not limited to, hydrophobic interaction
chromatography, ion exchange chromatography, affinity
chromatography, metal binding, immunoaffinity chromatography, and
high performance liquid chromatography or high pressure liquid
chromatography. Electrophoresis may comprise denaturing
electrophoresis or non-denaturing electrophoresis.
[0172] Antibodies or intermediates may comprise one or more tags.
The linkers may comprise one or more tags. The tags may be used to
purify the antibodies or intermediates. The one or more tags may be
cleaved by one or more proteases. Examples of tags include, but are
not limited to, polyhistidine, FLAG, HA, c-myc, V5, chitin binding
protein (CBP), maltose binding protein (MBP), and
glutathione-S-transferase (GST).
[0173] The methods may further comprise lyophilization or
ultracentrifugation of the antibodies or intermediates.
[0174] The purity of the bispecific antibody may be equal to or
greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more. The purity of the
antibody may be equal to or greater than 85%. The purity of the
antibody may be equal to or greater than 90%. The purity of the
antibody may be equal to or greater than 95%. The purity of the
antibody may be equal to or greater than 97%.
[0175] The purity of the intermediate (e.g., antibody-linker,
antibody fragment-linker,) may be equal to or greater than 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more. The purity of the antibody may be equal
to or greater than 85%. The purity of the antibody may be equal to
or greater than 90%. The purity of the antibody may be equal to or
greater than 95%. The purity of the antibody may be equal to or
greater than 97%.
[0176] The homogeneity of the bispecific antibody may be equal to
or greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. The homogeneity of
the antibody may be equal to or greater than 85%. The homogeneity
of the antibody may be equal to or greater than 90%. The
homogeneity of the antibody may be equal to or greater than 95%.
The homogeneity of the antibody may be equal to or greater than
97%.
[0177] The homogeneity of the intermediate (e.g., antibody-linker,
antibody fragment-linker,) may be equal to or greater than 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more. The homogeneity of the antibody may be
equal to or greater than 85%. The homogeneity of the antibody may
be equal to or greater than 90%. The homogeneity of the antibody
may be equal to or greater than 95%. The homogeneity of the
antibody may be equal to or greater than 97%.
VI. Cells
[0178] The bispecific antibodies disclosed herein may bind to one
or more receptors, co-receptors, antigens, or cell markers on one
or more cells. The bispecific antibodies disclosed herein may
comprise (a) an anti-CD3 antibody or antibody fragment and (b) a
second antibody or antibody fragment, wherein (i) the anti-CD3
antibody or antibody fragment binds to or interacts with CD3 on a
first cell; (ii) the second antibody or antibody fragment binds to
or interacts with a receptor, co-receptor, antigen or cell marker
on a second cell; or (iii) a combination of (i) and (ii), and
wherein (iv) the anti-CD3 antibody or antibody fragment comprises
one or more unnatural amino acids; (v) the second antibody or
antibody fragment comprises one or more unnatural amino acids; or
(vi) a combination of (iv) and (v). The first cell may be an
effector cell. The effector cell may be a T cell. The second cell
may be a target cell. The target cell may be a cancerous cell. The
first cell and the second cell may be the same type of cell. The
first cell and the second cell may be different cell types.
Alternatively, the first cell and the second cell may be the same
cell.
[0179] The one or more cells may be hematopoietic cells.
Hematopoietic cells include, but are not limited to, basophilic
myelocytes, basophils, B-cells, burst forming unit erythroid
(BFU-E), burst forming unit megakaryocytes (BFU-Mk), colony forming
unit basophils (CFU-Bas), colony forming unit erythroid (CFU-E),
colonly forming unit eosinophils (CFU-Eo), colony forming unit
granulocytes (CFU-G), colony forming unit granulocyte erythrocyte
monocyte macrophage (CFU-GEMM), colony forming unit granulocyte
maccrophage (CFU-GM), colony forming unite megakaryocyte (CFU-Mk,
CFU-MEG), common dendritic progenitor, common lymphoid progenitor
cells, common myeloerythroid progenitors, common myeloid
progenitors, common myelolymphoid progenitors, double negative 1
(DN1) cells, DN2 cells, DN3 cells, DN4 cells, double-positive cells
(DP cells), eosinophilic myelocytes, eosinophils, erythrocytes,
lymphoid stem cells, lymphoid-related dendritic cells, macrophages,
mast cells, megakaryocytes, memory B-cells, memory cells, memory
T-cells, monoblasts, monocytes, myeloblasts, myeloid stem cells,
myeloid-related dendritic cells, neutrophilic myelocytes,
neutrophils, natural killer cells (NK-cells), natural killer
T-cells (NKT-cells), platelets, plasma cells, plasma B cells,
plasmocytes, effector B cells, mature B cells, pro-B1-cells,
pro-B-2-cells, pro-B-cells, proerythroblasts, promonocytes,
regulatory T-cells (Tregs), T-cells, T-helper (Th) cells, Th0
cells, Th1 cells, Th2 cells, Th3 cells, Th17 cells. BFU-E or CFU-E
may refer to erythroid precursor cells that may differentiate into
erythrocytes. CFU-E cells may be more developed than BFU-E cells.
CFU-Eo may refer to developmental type of blood-forming cells that
may develop into eosinophils. CFU-G may refer to a developmental
type of blood-forming cells that may be a precursor of
granulocytes. CFU-GEMM may refer to a pluripotent type of precursor
cell in the lineage of blood-forming cells that may differentiate
into granuclocytes, erythrocytes, monocytes and/or macrophages.
CFU-GM may refer to a pluropotent type of precursor cell in the
lineage of blood-forming cells that may differentiate into
granuloctyes and/or macrophages. BFU-Mk, CFU-Mk or CFU-MEG may
refer to precursor cells that may differentiate into
megakaryocytes. CFU-Mk or CFU-MEG cells may be more developed than
BFU-Mk cells.
[0180] The one or more cells may be from an organ or tissue. The
organ may be a gland organ. The organ may be an organ of the
digestive or endocrine system. The organ may be both an endocrine
gland and a digestive organ. The organ may be derived from
endoderm, ectoderm, primitive endoderm, or mesoderm. The organ may
be an adrenal gland. In some cases, the adrenal gland comprises
chromaffin cells or ganglion cells. Alternatively, the organ is an
appendix, bladder, or brain. In some cases, the brain comprises
neurons (e.g., nerve cells) or glial cells. Glial cells include,
but are not limited to, astrocytes, oligodendrocytes, and ependymal
cells. In some instances, the organ is an ear, esophagus, eye, or
gallbladder. The gallbladder comprises cholecystocytes. The organ
may be a kidney. The kidney may comprise a kidney glomerulus
parietal cell, kidney glomerululs podocyte, kidney proximal tubule
brush border cell, Loop of Henle thin segment cell, thick ascending
limb cell, kidney distal tubule cell, kidney collecting ductal
cell, or interstitial kidney cell. In some instances, the organ is
a large intestine and the large intestine may comprise enterocytes,
goblet cells, caveolated tuft cells, enteroendocrine cells, or
ganglion neurons. The organ may be a liver. The liver may comprise
parenchymal or non-parenchymal cells. Examples of parenchymal cells
comprise hepatocytes. Non-parenchymal cells include, but are not
limited to, sinusoidal endothelial cells, kupffer cells and hepatic
stellate cells. In some instances, the organ is a lung, mouth,
nose, parathyroid gland, pineal gland, pituitary gland, skin, small
intestine, stomach, spleen, thymus, thyroid gland, trachea, uterus,
or vermiform appendix. In some instances, the organ may be a heart.
In some instances, the heart comprises cardiomyocytes. In some
instances, the organ is a muscle (e.g., heart muscle, skeletal
muscle, smooth muscle, etc.). The muscle may comprise myocytes.
[0181] In some cases, the cells are from a tissue. The tissue may
be a connective tissue, epithelial tissue, muscular tissue, or
nervous tissue. Alternatively, the tissue is a bone, tendon (both
referred to as musculoskeletal grafts), cornea, skin, heart valve,
or vein.
[0182] Connective tissue may be a fibrous tissue and is often found
throughout the body. Examples of connective tissues include, but
are not limited to, connective tissue, fat tissue, dense fibrous
tissue, cartilage, bone, blood, and lymph. Generally, connective
tissue has three main components: cells, fibers, and extracellular
matrix, which may be embedded in the body fluids. Fibroblasts are
often the cells responsible for the production of connective
tissue. The interaction of the fibers, the extracellular matrix and
the water, together, may form the pliable connective tissue as a
whole. Connective tissue may make up a variety of physical
structures including tendons and the connective framework of fibers
in muscles, capsules and ligaments around joints, cartilage, bone,
adipose tissue, blood and lymphatic tissue. Connective tissue (CT)
may be classified into three subtypes: embryonic CT, proper CT, and
special CT. The proper CT subtype may include dense regular CT,
dense irregular CT, and loose CT. The special CT subtype may
include cartilage, bone, adipose tissue, blood, hematopoietic
tissue and lymphatic tissue.
[0183] Often connective tissues have distinct functions,
characteristics, and compositions. The functions of connective
tissue may include storage of energy, protection of organs,
providing structural framework for the body, and connection of body
tissues. The connective tissue may be characterized by cells that
are spread through an extracellular fluid. In some instances, the
connective tissue may comprise a ground substance, which is often a
clear, colorless, and viscous fluid containing glycosaminoglycans
and proteoglycans. The ground substance may fix the bodywater and
the collagen fibers in the intercellular spaces. Ground substance
may also slow the spread of pathogens.
[0184] The connective tissue may be fibrous and the fibrous tissue
may comprise distinct compositions and be localized to specific
areas of the body. For example, collagenous fibers often contain
alpha polypeptide chains and may be primarily localized to a
tendon, ligament, skin, cornea, cartilage, bone, blood vessels,
gut, and intervertebral disc. In another example, elastic fibers
may comprise elastic microfibrill and elastin and may be primarily
localized to an extracellular matrix. Reticular fibers are another
example of fibrous tissue and may be localized to the liver, bone
marrow, or lymphatic organs.
[0185] However, not all types of connective tissues are fibrous.
Examples of non-fibrous connective tissues are adipose tissue and
blood. Adipose tissue may provide a "mechanical cushioning" to our
body. Although there is often no dense collagen network in adipose
tissue, groups of adipose cells may be kept together by collagen
fibers and collagen sheets in order to keep fat tissue under
compression in place (for example the sole of the foot).
[0186] Epithelia are tissues that may consist of closely apposed
cells without intervening intercellular substances. Epithelia are
often avascular, but epithelia may "grow" on an underlying layer of
vascular connective tissue. The connective tissue and the
epithelium may be separated by a basement membrane. Epithelium may
cover all free surfaces of the body. Epithelium may also line the
large internal body cavities, where it is termed mesothelium.
Furthermore, the internal surfaces of blood and lymph vessels may
be lined by epithelium, here called endothelium. Epithelia are
often classified on the basis of the number of cell layers and the
shape of the cells in the surface layer. If there is only one layer
of cells in the epithelium, it is designated simple. If there are
two or more layers of cells, it is termed stratified. Cells in the
surface layer may be described according to their height as
squamous (scale- or plate-like), cuboidal or columnar.
[0187] Different types of epithelial tissues may have specialized
functions and locations within the body. For example,
pseudostratified columnar may function to remove dust and particles
from airways and may have cilia. The pseudostratified columnar may
line the respiratory passageways. The simple columnar may be
involved in absorption and often line the uterus and most organs of
the digestive tract. The simple cuboidal may be involved in
secretion and absorption and may be localized to glands, kidney
tubules, and ovaries. The simple squamous may play a role in
diffusion and filtration and may be localized to lungs, walls of
capillaries and vessels. The stratified squamous may protect
underlying cells and is often localized to the skin, throat,
vagina, and mouth. The stratified cuboidal may be involved in
protection and may line ducts of the mammary glands, sweat glands,
and pancreas. The stratified columnar may be involved in protection
and secretion and may be localized to the male urethra and vas
deferens, and parts of the pharynx.
[0188] Muscular tissue is often a contractile tissue and may be
derived from the mesodermal layer of embryonic germ cells. Muscle
cells may contain contractile filaments that move past each other
and change the size of the cell. They are classified as skeletal,
cardiac, or smooth muscles. Skeletal muscle or "voluntary muscle"
may be anchored by tendons (or by aponeuroses at a few places) to
bone and may be used to effect skeletal movement such as locomotion
and in maintaining posture. Smooth muscle or "involuntary muscle"
is often found within the walls of organs and structures such as
the esophagus, stomach, intestines, bronchi, uterus, urethra,
bladder, blood vessels, and the arrector pili in the skin (in which
it controls erection of body hair). Cardiac muscle is also an
"involuntary muscle" but may be more structurally similar to
skeletal muscle, and is often found in the heart.
[0189] Cardiac and skeletal muscles are often "striated" in that
they contain sarcomeres and are packed into highly regular
arrangements of bundles. While skeletal muscles may be arranged in
regular, parallel bundles, cardiac muscle often connects at
branching, irregular angles (called intercalated discs). Striated
muscle may contract and relax in short, intense bursts, whereas
smooth muscle may sustain longer or even near-permanent
contractions.
[0190] Skeletal muscle may be divided into several subtypes. Type
I, slow oxidative, slow twitch, or "red" muscle is often dense with
capillaries and may be rich in mitochondria and myoglobin, giving
the muscle tissue its characteristic red color. It may carry more
oxygen and sustain aerobic activity. Type II, fast twitch muscle,
has three major kinds, Type IIa, Type IIx, and Type IIb. Type IIa
is often aerobic and may be rich in mitochondria and capillaries
and may appear red. Type IIx (also known as type IId), which is
often less dense in mitochondria and myoglobin. Type IIb, which may
be anaerobic, glycolytic, "white" muscle that is often even less
dense in mitochondria and myoglobin.
[0191] Nervous tissue is one of four major classes of tissue.
Nervous tissue is often the main component of the nervous system,
the brain, spinal cord, and nerves, which may regulate and control
body functions. Nervous tissue is often composed of neurons and the
neuroglia cells. Neurons may transmit impulses. Neuroglial cells
may assist in propagation of the nerve impulse as well as provide
nutrients to the neuron. Nervous tissue is often made of nerve
cells that may come in many varieties, all of which may be
distinctly characteristized by the axon or long stem like part of
the cell that sends action potential signals to the next cell.
[0192] Functions of the nervous system may include sensory input,
integration, controls of muscles and glands, homeostasis, and
mental activity. Nervous tissue may react to stimuli and may
conduct impulses to various organs in the body which often bring
about a response to the stimulus. Nerve tissue (as in the brain,
spinal cord and peripheral nerves that branch throughout the body)
are often made up of specialized nerve cells called neurons.
Neurons are easily stimulated and transmit impulses very rapidly. A
nerve often comprises many nerve cell fibers (neurons) bound
together by connective tissue. A sheath of dense connective tissue,
the epineurium may surround the nerve. This sheath penetrates the
nerve to form the perineurium which surrounds bundles of nerve
fibers. Blood vessels of various sizes may be seen in the
epineurium. The endoneurium, which consists of a thin layer of
loose connective tissue, surrounds the individual nerve fibers.
[0193] The cell body may be enclosed by a cell (plasma) membrane
and may have a central nucleus. Granules called Nissl bodies are
often found in the cytoplasm of the cell body. Within the cell
body, extremely fine neurofibrils may extend from the dendrites
into the axon. The axon is often surrounded by the myelin sheath,
which forms a whitish, non-cellular, fatty layer around the axon.
Outside the myelin sheath may be a cellular layer called the
neurilemma or sheath of Schwann cells. The myelin sheath together
with the neurilemma is also known as the medullary sheath. This
medullary sheath may be interrupted at intervals by the nodes of
Ranvier.
[0194] Neurons may be classified both structurally and
functionally. Structural classification may group neurons according
to the number of processes extending from their cell body. Three
major neuron groups often make up this classification: multipolar
(polar=end, pole), bipolar and unipolar neurons. Multipolar neurons
often have three or more processes. These are the most common
neuron type in humans (more than 99% of neurons belong to this
class) and the major neuron type in the CNS. Bipolar neurons are
often spindle-shaped, with a dendrite at one end and an axon at the
other. An example may be found in the light-sensitive retina of the
eye. Unipolar neurons often comprise sensory neurons. Sensory
neurons normally have only a single process or fibre which divides
close to the cell body into two main branches (axon and
dendrite).
[0195] The cells may also comprise hair follicles, hair cells, ear
hair cells, ear hair stem cells, or cochlear cells. Hair cells are
often the sensory receptors of both the auditory system and the
vestibular system. The auditory hair cells may be located within
the organ of Corti on a thin basilar membrane in the cochlea of the
inner ear. Cochlear hair cells may come in two anatomically and
functionally distinct types: the outer and inner hair cells.
[0196] The cells described herein may be diseased, infected,
mutated or genetically modified.
[0197] The one or more cells may be a pathogenic cell. Pathogenic
cells include, but are not limited to, bacteria, viruses, fungi,
and protozoans. Examples of pathogens may include, but are not
limited to, the bacteria, viruses, fungi, and protozoans disclosed
herein.
VII. Receptors, Co-Receptors, Antigens and Cell Markers
[0198] The bispecific antibodies disclosed herein may bind to one
or more receptors, co-receptors, antigens, or cell markers on one
or more cells. The bispecific antibodies disclosed herein may
comprise (a) a anti-CD3 antibody or antibody fragment and (b) a
second antibody or antibody fragment, wherein (i) the anti-CD3
antibody or antibody fragment binds to or interacts with CD3 on a
first cell; (ii) the second antibody or antibody fragment binds to
or interacts with a receptor, co-receptor, antigen or cell marker
on a second cell; or (iii) a combination of (i) and (ii), and
wherein (iv) the anti-CD3 antibody or antibody fragment comprises
one or more unnatural amino acids; (v) the second antibody or
antibody fragment comprises one or more unnatural amino acids; or
(vi) a combination of (iv) and (v). The first cell and the second
cell may be the same type of cell. The first cell and the second
cell may be different cell types. Alternatively, the first cell and
the second cell may be the same cell.
[0199] For example, for a bispecific antibody of Formula I, IA, II,
and/or HA, X may comprise an antibody or antibody fragment that
binds to CD3 or a portion thereof. Alternatively, or additionally,
for an antibody of Formula I, IA, II, and/or IIA, Y comprises an
antibody or antibody fragment that may bind to a receptor,
co-receptor, antigen, transmembrane protein or cell marker.
[0200] Examples of receptors, co-receptors, antigens or cell
markers may include, but are not limited to, a receptor,
co-receptor, antigen or cell marker on a hematopoietic cell, tissue
cell, epithelial cell, mesothelial cell, dermal cell, endothelial
cell, dendritic cell, vascular cell, stromal cell, neuron, cancer
cell, bacteria, fungus, or virus. The receptors, co-receptors,
antigens or cell markers may be selected from the group comprising
CD19, CD20, CD22, CD25, CD30, CD40, CD56, CD64, CD70, CD74, CD79,
CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB,
PSMA, Cripto, ED-8, TMEFF2, EphB2, EphA2, FAP, av integrin,
mesothelin, EGFR, TAG-72, GD2, CAIX, and 5T4.
[0201] An antigen may evoke the production of one or more
antibodies. An antigen may refer to a molecule or molecular
fragment that may be bound by a major histocompatiblity complex
(MHC) and presented to a T-cell receptor. The term "antigen" may
also refer to an immunogen. An immunogen may provoke an adaptive
immune response if injected on its own into a subject. An immunogen
may induce an immune response by itself. An antigen may also refer
to a hapten. A hapten may be a small molecule. Generally, a hapten
may induce an immune repsonse when attached to a larger carrier
molecule, such as a protein. Antigens may be proteins or
polysaccharides. Antigens may comprise parts (e.g., coats,
capsules, cell walls, flagella, fimbrae, and toxins) of bacteria,
viruses, and other microorganisms. Lipids and nucleic acids may be
antigenic when combined with proteins and polysaccharides. Antigens
may include superantigens, T-dependent antigens and T-independent
antigens.
[0202] Antigens may be exogenous antigens or endogeneous antigens.
Exogeneous antigens are typically antigens that have entered the
body from the outside, for example by inhalation, ingestion, or
injection. Some antigens may start out as exogenous antigens, and
later become endogenous (for example, intracellular viruses).
Intracellular antigens may be released back into circulation upon
the destruction of the infected cell, again. Endogenous antigens
may be antigens that have been generated within previously-normal
cells as a result of normal cell metabolism, or because of viral or
intracellular bacterial infection.
[0203] Antigens may also include autoantigens. An autoantigen may
be a normal protein or complex of proteins (and sometimes DNA or
RNA) that is recognized by the immune system of patients suffering
from a specific autoimmune disease. These antigens should, under
normal conditions, not be the target of the immune system, but, due
to mainly genetic and environmental factors, the normal
immunological tolerance for such an antigen has been lost in these
patients.
[0204] Antigens may include tumor antigens. Tumor antigens or
neoantigens may be antigens that are presented by MHC I or MHC II
molecules on the surface of tumor cells. These antigens may
sometimes be presented by tumor cells and never by the normal ones.
In this case, they are called tumor-specific antigens (TSAs) and,
in general, result from a tumor-specific mutation. More common are
antigens that are presented by tumor cells and normal cells, and
they are called tumor-associated antigens (TAAs). Cytotoxic T
lymphocytes that recognize these antigens may be able to destroy
the tumor cells before they proliferate or metastasize. Tumor
antigens may also be on the surface of the tumor in the form of,
for example, a mutated receptor, in which case they may be
recognized by B cells.
VIII. Indications
[0205] Disclosed herein are methods for treating a disease or
condition in a subject in need thereof, comprising administering
the bispecific antibodies disclosed herein.
[0206] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody of Formula
I, IA, II or IIA. The condition or disease may comprise a cancer.
The cancer may be selected from a prostate cancer, a myeloid
leukemia, an epithelial cancer, an ovarian cancer, a cervical
cancer, a breast cancer, a lung cancer, a kidney cancer, a
colorectal cancer, a brain cancer, a thyroid cancer, an
astrocytoma, a pancreatic cancer, an endometrial cancer, a
neuroendocrine cancer, a gastroenteropancreatic tumor, a
non-Hodgkin's lymphoma, a skin cancer, a gastric cancer, a
glioblastoma and an Ewing's sarcoma. The lung cancer may be a small
cell lung cancer. The ovarian cancer may be a stromal ovarian
cancer. The thyroid cancer may be a medullary thyroid cancer. The
skin cancer may be a melanoma. The skin cancer may be a
neo-angiogenic skin cancer. The pancreatic cancer may be an
exocrine pancreatic cancer.
[0207] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody comprising
an anti-CD3 antibody or fragment thereof conjugated to an anti-Her2
antibody or fragment thereof to a subject in need thereof. The
condition or disease may be a cancer. The cancer may be a breast
cancer.
[0208] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody comprising
an anti-CD3 antibody or fragment thereof conjugated to an
anti-EGFRvIII antibody or fragment thereof to a subject in need
thereof. The condition or disease may be a cancer. The cancer may
be a glioma or a glioblastoma.
[0209] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody comprising
an anti-CD3 antibody or fragment thereof conjugated to an anti-CS1
antibody or fragment thereof to a subject in need thereof. The
condition or disease may be a cancer. The cancer may be a multiple
myeloma.
[0210] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody comprising
an anti-CD3 antibody or fragment thereof conjugated to an
anti-CLL-1 antibody or fragment thereof to a subject in need
thereof. The condition or disease may be a cancer. The cancer may
be an acute myeloid leukemia.
[0211] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody comprising
an anti-CD3 antibody or fragment thereof conjugated to an anti-CD33
antibody or fragment thereof to a subject in need thereof. The
condition or disease may be a cancer. The cancer may be an acute
myeloid leukemia.
[0212] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody comprising
an anti-CD3 antibody or fragment thereof conjugated to an anti-ROR1
antibody or fragment thereof to a subject in need thereof. The
condition or disease may be a cancer. The cancer may be a B-cell
chronic lymphocytic leukemia. The cancer may be a mantle cell
lymphoma. The cancer may be an acute lymphoblastic leukemia.
[0213] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody comprising
an anti-CD3 antibody or fragment thereof conjugated to an
anti-CD44v6 antibody or fragment thereof to a subject in need
thereof. The condition or disease may be a cancer. The cancer may
be a B-cell chronic lymphocytic leukemia. The cancer may be a
mantle cell lymphoma. The cancer may be an acute myeloid
leukemia.
[0214] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody comprising
an anti-CD3 antibody or fragment thereof conjugated to an
anti-IL13R.alpha.2 antibody or fragment thereof to a subject in
need thereof. The condition or disease may be a cancer. The cancer
may be a glioblastoma.
[0215] Disclosed herein are methods of treating a disease or
condition comprising administering a bispecific antibody comprising
an anti-CD3 antibody or fragment thereof conjugated to a bscWue1
antibody or fragment thereof to a subject in need thereof. The
condition or disease may be a cancer. The cancer may be a multiple
myeloma.
[0216] The bispecific antibodies and compositions disclosed herein
may be used to treat one or more diseases or conditions in a
subject in need thereof. The one or more diseases or conditions may
be a cancer, a pathogenic infection, autoimmune disease,
inflammatory disease, or genetic disorder.
[0217] In some instances, the one or more diseases comprise a
cancer. The cancer may be a recurrent and/or refractory cancer.
Examples of cancers include, but are not limited to, sarcomas,
carcinomas, gliomas, lymphomas or leukemias. The cancer may be a
breast cancer. The cancer may be a prostate cancer. The cancer may
be a lung cancer. The cancer may be a glioma. The cancer may be a
glioblastoma. The cancer may be a myeloma. The cancer may be
multiple myeloma. The multiple myeloma may be a relapsed multiple
myeloma. The multiple myeloma may be refractory. The leukemia may
be acute myeloid leukemia. The leukemia may be B-cell chronic
lymphocytic leukemia. The leukemia may be mantle cell leukemia. The
leukemia may be acute lymphoblastic leukemia.
[0218] Sarcomas are cancers of the bone, cartilage, fat, muscle,
blood vessels, or other connective or supportive tissue. Sarcomas
include, but are not limited to, bone cancer, fibrosarcoma,
chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma,
malignant schwannoma, bilateral vestibular schwannoma,
osteosarcoma, soft tissue sarcomas (e.g. alveolar soft part
sarcoma, angiosarcoma, cystosarcoma phylloides,
dermatofibrosarcoma, desmoid tumor, epithelioid sarcoma,
extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma,
hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma,
neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma).
[0219] Carcinomas are cancers that begin in the epithelial cells,
which are cells that cover the surface of the body, produce
hormones, and make up glands. By way of non-limiting example,
carcinomas include breast cancer, pancreatic cancer, lung cancer,
colon cancer, colorectal cancer, rectal cancer, kidney cancer,
bladder cancer, stomach cancer, prostate cancer, liver cancer,
ovarian cancer, brain cancer, vaginal cancer, vulvar cancer,
uterine cancer, oral cancer, penile cancer, testicular cancer,
esophageal cancer, skin cancer, cancer of the fallopian tubes, head
and neck cancer, gastrointestinal stromal cancer, adenocarcinoma,
cutaneous or intraocular melanoma, cancer of the anal region,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, cancer of the urethra, cancer of the
renal pelvis, cancer of the ureter, cancer of the endometrium,
cancer of the cervix, cancer of the pituitary gland, neoplasms of
the central nervous system (CNS), primary CNS lymphoma, brain stem
glioma, and spinal axis tumors. In some instances, the cancer is a
skin cancer, such as a basal cell carcinoma, squamous, melanoma,
nonmelanoma, or actinic (solar) keratosis.
[0220] In some instances, the cancer is a lung cancer. Lung cancer
may start in the airways that branch off the trachea to supply the
lungs (bronchi) or the small air sacs of the lung (the alveoli).
Lung cancers include non-small cell lung carcinoma (NSCLC), small
cell lung carcinoma, and mesotheliomia. Examples of NSCLC include
squamous cell carcinoma, adenocarcinoma, and large cell carcinoma.
The mesothelioma may be a cancerous tumor of the lining of the lung
and chest cavitity (pleura) or lining of the abdomen (peritoneum).
The mesothelioma may be due to asbestos exposure. The cancer may be
a brain cancer, such as a glioblastoma.
[0221] In some instances, the cancer is a breast cancer. In some
instances, the breast cancer is estrogen receptor positive,
progesterone receptor positive and Her2 positive (triple positive).
In some instances, the breast cancer is estrogen receptor negative,
progesterone receptor negative and Her2 negative (triple negative).
In some instances, the breast cancer is estrogen receptor positive
and Her2 positive. In some instances, the breast cancer is estrogen
receptor positive and Her 2 negative. In some instances, the breast
cancer is estrogen receptor negative and Her2 positive. In some
instances, the breast cancer is metastatic.
[0222] Alternatively, the cancer may be a central nervous system
(CNS) tumor. CNS tumors may be classified as gliomas or nongliomas.
The glioma may be malignant glioma, high grade glioma, diffuse
intrinsic pontine glioma. Examples of gliomas include astrocytomas,
oligodendrogliomas (or mixtures of oligodendroglioma and astocytoma
elements), and ependymomas. Astrocytomas include, but are not
limited to, low-grade astrocytomas, anaplastic astrocytomas,
glioblastoma multiforme, pilocytic astrocytoma, pleomorphic
xanthoastrocytoma, and subependymal giant cell astrocytoma.
Oligodendrogliomas include low-grade oligodendrogliomas (or
oligoastrocytomas) and anaplastic oligodendriogliomas. Nongliomas
include meningiomas, pituitary adenomas, primary CNS lymphomas, and
medulloblastomas. In some instances, the cancer is a
meningioma.
[0223] The leukemia may be an acute lymphocytic leukemia, acute
lymphoblastic leukemia, acute myelocytic leukemia, chronic
lymphocytic leukemia, or chronic myelocytic leukemia. Additional
types of leukemias include hairy cell leukemia, chronic
myelomonocytic leukemia, B-cell chronic lymphocytic leukemia and
juvenile myelomonocytic leukemia.
[0224] Lymphomas are cancers of the lymphocytes and may develop
from either B or T lymphocytes. The two major types of lymphoma are
Hodgkin's lymphoma, previously known as Hodgkin's disease, and
non-Hodgkin's lymphoma. Hodgkin's lymphoma is marked by the
presence of the Reed-Sternberg cell. Non-Hodgkin's lymphomas are
all lymphomas which are not Hodgkin's lymphoma. Non-Hodgkin
lymphomas may be indolent lymphomas and aggressive lymphomas.
Non-Hodgkin's lymphomas include, but are not limited to, diffuse
large B cell lymphoma, follicular lymphoma, mucosa-associated
lymphatic tissue lymphoma (MALT), small cell lymphocytic lymphoma,
mantle cell lymphoma, Burkitt's lymphoma, mediastinal large B cell
lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell
lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), extranodal
marginal zone B cell lymphoma, intravascular large B cell lymphoma,
primary effusion lymphoma, and lymphomatoid granulomatosis.
[0225] The one or more diseases or conditions may be a pathogenic
infection. Pathogenic infections may be caused by one or more
pathogens. In some instances, the pathogen is a bacterium, fungi,
virus, or protozoan.
[0226] Exemplary pathogens include but are not limited to:
Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia,
Chlamydophila, Clostridium, Corynebacterium, Enterococcus,
Escherichia, Francisella, Haemophilus, Helicobacter, Legionella,
Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria,
Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus,
Streptococcus, Treponema, Vibrio, or Yersinia. In some cases, the
disease or condition caused by the pathogen is tuberculosis and the
heterogeneous sample comprises foreign molecules derived from the
bacterium Mycobacterium tuberculosis and molecules derived from the
subject. In some instances, the disease or condition is caused by a
bacterium is tuberculosis, pneumonia, which may be caused by
bacteria such as Streptococcus and Pseudomonas, a foodborne
illness, which may be caused by bacteria such as Shigella,
Campylobacter and Salmonella, and an infection such as tetanus,
typhoid fever, diphtheria, syphilis and leprosy. The disease or
condition may be bacterial vaginosis, a disease of the vagina
caused by an imbalance of naturally occurring bacterial flora.
Alternatively, the disease or condition is a bacterial meningitis,
a bacterial inflammation of the meninges (e.g., the protective
membranes covering the brain and spinal cord). Other diseases or
conditions caused by bacteria include, but are not limited to,
bacterial pneumonia, a urinary tract infection, bacterial
gastroenteritis, and bacterial skin infection. Examples of
bacterial skin infections include, but are not limited to, impetigo
which may be caused by Staphylococcus aureus or Streptococcus
pyogenes; erysipelas which may be caused by a streptococcus
bacterial infection of the deep epidermis with lymphatic spread;
and cellulitis which may be caused by normal skin flora or by
exogenous bacteria.
[0227] The pathogen may be a fungus, such as, Candida, Aspergillus,
Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Examples
of diseases or conditions caused by a fungus include, but are not
limited to, jock itch, yeast infection, ringworm, and athlete's
foot.
[0228] The pathogen may be a virus. Examples of viruses include,
but are not limited to, adenovirus, coxsackievirus, Epstein-Barr
virus, Hepatitis virus (e.g., Hepatitis A, B, and C), herpes
simplex virus (type 1 and 2), cytomegalovirus, herpes virus, HIV,
influenza virus, measles virus, mumps virus, papillomavirus,
parainfluenza virus, poliovirus, respiratory syncytial virus,
rubella virus, and varicella-zoster virus. Examples of diseases or
conditions caused by viruses include, but are not limited to, cold,
flu, hepatitis, AIDS, chicken pox, rubella, mumps, measles, warts,
and poliomyelitis.
[0229] The pathogen may be a protozoan, such as Acanthamoeba (e.g.,
A. astronyxis, A. castellanii, A. culbertsoni, A. hatchetti, A.
polyphaga, A. rhysodes, A. healyi, A. divionensis), Brachiola
(e.g., B. connori, B. vesicularum), Cryptosporidium (e.g., C.
parvum), Cyclospora (e.g., C. cayetanensis), Encephalitozoon (e.g.,
E. cuniculi, E. hellem, E. intestinalis), Entamoeba (e.g., E.
histolytica), Enterocytozoon (e.g., E. bieneusi), Giardia (e.g., G.
lamblia), Isospora (e.g, I. belli), Microsporidium (e.g., M.
africanum, M. ceylonensis), Naegleria (e.g., N. fowleri), Nosema
(e.g., N. algerae, N. ocularum), Pleistophora, Trachipleistophora
(e.g., T. anthropophthera, T. hominis), and Vittaforma (e.g., V.
corneae).
[0230] The disease or condition may be an autoimmune disease or
autoimmune related disease. An autoimmune disorder may be a
malfunction of the body's immune system that causes the body to
attack its own tissues. Examples of autoimmune diseases and
autoimmune related diseases include, but are not limited to,
Addison's disease, alopecia areata, ankylosing spondylitis,
antiphospholipid syndrome (APS), autoimmune aplastic anemia,
autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune
myocarditis, Behcet's disease, celiac sprue, Crohn's disease,
dermatomyositis, eosinophilic fasciitis, erythema nodosum, giant
cell arteritis (temporal arteritis), Goodpasture's syndrome,
Graves' disease, Hashimoto's disease, idiopathic thrombocytopenic
purpura (ITP), IgA nephropathy, juvenile arthritis, diabetes,
juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, lupus
(SLE), mixed connective tissue disease (MCTD), multiple sclerosis,
myasthenia gravis, pemphigus, polyarteritis nodosa, type I, II,
& III autoimmune polyglandular syndromes, polymyalgia
rheumatica, polymyositis, psoriasis, psoriatic arthritis, Reiter's
syndrome, relapsing polychondritis, rheumatoid arthritis,
sarcoidosis, scleroderma, Sjogren's syndrome, sperm &
testicular autoimmunity, stiff person syndrome, Takayasu's
arteritis, temporal arteritis/giant cell arteritis, ulcerative
colitis, uveitis, vasculitis, vitiligo, and Wegener's
granulomatosis.
[0231] The disease or condition may be an inflammatory disease.
Examples of inflammatory diseases include, but are not limited to,
alveolitis, amyloidosis, angiitis, ankylosing spondylitis,
avascular necrosis, Basedow's disease, Bell's palsy, bursitis,
carpal tunnel syndrome, celiac disease, cholangitis, chondromalacia
patella, chronic active hepatitis, chronic fatigue syndrome,
Cogan's syndrome, congenital hip dysplasia, costochondritis,
Crohn's Disease, cystic fibrosis, De Quervain's tendinitis,
diabetes associated arthritis, diffuse idiopathic skeletal
hyperostosis, discoid lupus, Ehlers-Danlos syndrome, familial
mediterranean fever, fascitis, fibrositis/fibromyalgia, frozen
shoulder, ganglion cysts, giant cell arteritis, gout, Graves'
Disease, HIV-associated rheumatic disease syndromes,
hyperparathyroid associated arthritis, infectious arthritis,
inflammatory bowel syndrome/irritable bowel syndrome, juvenile
rheumatoid arthritis, lyme disease, Marfan's Syndrome, Mikulicz's
Disease, mixed connective tissue disease, multiple sclerosis,
myofascial pain syndrome, osteoarthritis, osteomalacia,
osteoporosis and corticosteroid-induced osteoporosis, Paget's
Disease, palindromic rheumatism, Parkinson's Disease, Plummer's
Disease, polymyalgia rheumatica, polymyositis, pseudogout,
psoriatic arthritis, Raynaud's Phenomenon/Syndrome, Reiter's
Syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis,
sciatica (lumbar radiculopathy), scleroderma, scurvy, sickle cell
arthritis, Sjogren's Syndrome, spinal stenosis, spondyloisthesis,
Still's Disease, systemic lupus erythematosis, Takayasu's
(Pulseless) Disease, Tendinitis, tennis elbow/golf elbow, thyroid
associated arthritis, trigger finger, ulcerative colitis, Wegener's
Granulomatosis, and Whipple's Disease.
IX. Immune Modulation
[0232] The antibodies disclosed herein may be used to modulate an
immune response. Modulation of an immune response may comprise
stimulating, activating, increasing, enhancing, or up-regulating an
immune response. Modulation of an immune response may comprise
suppressing, inhibiting, preventing, reducing, or downregulating an
immune response. For example, the antibodies may comprise an
anti-CD3 antibody or antibody fragment that may bind to a first
cell and a second antibody or antibody fragment that may bind to a
second cell. Binding of the antibody to the first and second cell
may result in modulation of an immune response. The first cell may
be an immune cell. The immune cell may be a hematopoietic cell. The
second cell may be an immune cell, healthy cell, cancer cell,
bacteria, or virally-infected.
Examples
[0233] The following specific and non-limiting examples are to be
construed as merely illustrative, and do not limit the present
disclosure in any way whatsoever. Without further elaboration, it
is believed that one skilled in the art can, based on the
description herein, utilize the present disclosure to its fullest
extent. All patents, patent applications, and publications cited
herein are hereby incorporated by reference in their entirety.
Example 1
Expression of Anti-CD3 Fab Double Mutant in E. coli
[0234] UCHT1-Fab sequences were obtained from the literature and
inserted into a pBAD vector behind the stII signal sequence. The
sites for unnatural amino acid incorporation (light chain threonine
109 (LC-Thr109), light chain serine 202 (LC-Ser202), heavy chain
alanine 123 (HC-A123), and heavy chain lysine 138 (HC-Lys138)) were
quickchanged (QuikChange.RTM. Stratagene) to TAG amber nonsense
codon. For the double mutant, two residues (LC-Ser202 and
HC-Lys138) were mutated to TAG amber nonsense codon. FIG. 1 depicts
a ribbon diagram of the UCHT1-Fab fragment. The pBAD vector was
co-transformed with pULTRA-pAcF (a vector containing orthogonal M.
jannaschii tRNA and aminoacyl-tRNA synthetase specific for pAcF) in
DH10B cells. The cells were grown in LB media (2 L) supplemented
with 100 mg/ml ampicillin, 25 mg/ml chloramphenicol, and 1 mM pAcF
at 37.degree. C. and 250 rpm. At OD.sub.600 0.8, cells were induced
with 0.2% arabinose and moved to 30.degree. C. for 20 hours at 270
rpm. Cells were harvested and proteins were extracted by incubating
with 150 mL of periplasmic lysis buffer (20% sucrose, 30 mM Tris,
pH 8, 1 mM EDTA, and 0.2 mg/mL lysozyme) for 30 min at 37.degree.
C. Extracts were clarified by centrifugation (18000 rpm, 30 min),
filtered through 0.22 micron filter, and loaded onto Protein G
column (GE healthcare). The column was washed with 20 bed volumes
of 50 mM NaOAc, pH 5.2, and proteins were eluted with 10 bed
volumes of 100 mM glycine, pH 2.8. The eluents were immediately
neutralized by adding 10% of 1 M Tris, pH 8, and dialysed against
PBS for long-term storage.
Example 2
Synthesis of Bispecific Antibodies Using Genetically Encoded
Unnatural Amino Acids
[0235] Our strategy takes advantage of genetically encoded
unnatural amino acids with orthogonal chemical reactivity relative
to the canonical twenty amino acids to site-specifically modify
antibody fragments. Specifically, we used an evolved
tRNA/aminoacyl-tRNA synthetase pair to site-specifically
incorporate p-acetylphenylalanine (pAcF, FIG. 2A) at defined sites
in each of two Fab fragments in response to an amber nonsense
codon. The mutant Fab fragments were then selectively coupled by
stable oxime formation using the alkoxy-amine termini of two
bifunctional linkers (FIG. 2B). In a second step, the two
Fab-linker conjugates were linked to each other in order to obtain
the heterodimer through a copper-free [3+2] Huisgen-cycloaddition
("Click" reaction) (FIG. 2C). This approach has a number of
advantages over recombinant technologies and conventional coupling
chemistries. For example, the use of bioorthogonal chemistries
produces homogeneous, chemically-defined products; variable linker
lengths and conjugation sites on the antibody may be easily
optimized to ensure flexibility and good efficacy for each specific
application; sequences from existing monoclonal antibodies may be
directly adopted; and the modular approach easily and rapidly
allows for combinatorial generation of diverse heterodimers
(antibodies, enzymes, cytokines, etc.)
[0236] The bifunctional linkers (50-fold molar equivalents) were
then coupled to the pAcF-containing anti-HER2 Fab (5 mg/mL) in
excellent yields (>95% by ESI-MS, FIG. 9A-J) in 100 mM acetate
buffer pH 4.5 at 37.degree. C. for 16 hours. FIG. 3A-E depict
ESI-MS analysis of Fab fragments before linker and after linker
conjugation. FIG. 3A depicts Herceptin Fab mutant (LS202X, X=pAcF);
FIG. 3B depicts Herceptin Fab-(PEG).sub.4-Az; FIG. 9C depicts
Herceptin Fab-(PEG).sub.4-Octyne; FIG. 3D depicts UCHT1 Fab mutant
(HK138X, X=pAcF); and FIG. 3E depicts UCHT1 Fab-(PEG).sub.4-Octyne.
FIG. 3F-J depict deconvoluted mass spectrum of Fab fragments before
and after linker conjugation. FIG. 3F depicts Herceptin Fab mutant
(LS202X, X=pAcF); FIG. 3G depicts Herceptin Fab-(PEG).sub.4-Az;
FIG. 3H depicts Herceptin Fab-(PEG).sub.4-Octyne; FIG. 3I depicts
UCHT1 Fab mutant (HK138X, X=pAcF); and FIG. 3J depicts UCHT1
Fab-(PEG).sub.4-Octyne.
[0237] Excess linker was removed by an Amicon 10K concentrator
column (Millipore) or by size exclusion chromatography
(Superdex-75, GE Healthcare). The two Fab-linker conjugates were
separately buffer exchanged into PBS, pH 7.4, then mixed at a 1:1
ratio at 10 mg/mL and incubated at 37.degree. C. for the
copper-free Click reaction. The reaction was monitored by SDS-PAGE,
and a band at .about.100 kDa was observed, corresponding to the
molecular weight of the Fab dimer. After 48 hours, about 70% of
starting material was consumed, and the homodimer was easily
purified by size-exclusion chromatography (Superdex-200, GE
Healthcare) from the unreacted Fabs.
Example 3
Synthesis of Anti-Her2/Anti-CD3 Heterodimer
[0238] pAcPhe was substituted at HC-K138 in the anti-anti-CD3
antibody, UCHT1. This site is distal to the antigen binding site
and, when conjugated to the LC-5202 mutant anti-HER2 Fab with the
same polyethylene glycol linker used above, should be long and
flexible enough to allow the resulting bispecific antibody to
productively bind both a CD3 positive T-cell and the HER2 positive
target cell simultaneously. UCHT1 Fab was expressed in E. coli by
the same method as described for anti-HER2 Fab and in Example 2,
and the cyclooctyne linker-modified anti-CD3 was prepared as
described in Example 7 in >95% yield as confirmed by ESI-MS
(FIG. 3E, 3J). The anti-CD3-Fab-cyclooctyne conjugate was then
coupled to anti-HER2-Fab-azide conjugate as described in Example 7
in 70% percent yield (determined by SDS-PAGE and chromatographic
separation) and purified from unreacted Fab monomers by
Superdex-200 size exclusion column. FIG. 4A shows the SDS-PAGE of
the anti-HER2/anti-CD3 heterodimer. FIG. 4B shows the size
exclusion chromatography FPLC trace of the anti-HER2/anti-CD3
heterodimer.
Example 4
Binding Characterization of Anti-Her2/Anti-CD3 Heterodimer
[0239] FACS analysis of the Fab heterodimer showed affinity for
both HER2-positive (SK-BR-3) and CD3-positive (Jurkat) cells with
no cross-reactivity of anti-CD3 Fab and anti-HER2 Fab, respectively
(FIG. 5A-B). FIG. 5A shows the FACS analysis of CD3-positive
(Jurkat) cells treated with anti-k-PE (line 1), anti-CD3 Fab (line
2), anti-Her2 Fab (line 3) or anti-Her2/anti-CD3 heterodimer (line
4). FIG. 5B shows the FACS analysis of HER2-positive (SK-BR-3)
cells treated with anti-k-PE (line 1), anti-CD3 Fab (line 2),
anti-Her2 Fab (line 3) or anti-Her2/anti-CD3 heterodimer (line
4).
[0240] To demonstrate the simultaneous binding of the heterodimer
to the two antigens, we visualized cross-linking of fluorescently
labeled SK-BR-3 and Jurkat cells. Specifically, SK-BR-3 cells and
Jurkat cells were first stained with Mito Tracker Red (Invitrogen)
and carboxyfluorescein succinimidyl ester (CFSE, Invitrogen),
respectively. The labeled Jurkat cells were incubated with 100 nM
of the anti-HER2/anti-CD3 heterodimer in RPMI media supplemented
with 10% FBS (fetal bovine serum) at 4.degree. C. for 30 minutes,
and excess conjugate was washed away before mixing the Jurkat cells
with SK-BR3 cells. A 1:1 mixture of unconjugated anti-CD3 and
anti-HER2 Fabs was used as a negative control under the same
labeling conditions. The cells were incubated at 37.degree. C. for
6 hours, allowing the adherent SK-BR-3 cells to attach to the
plate; excess Jurkat cells were removed by gentle washing with
media. In the sample with the heterodimer, significantly more
Jurkat cells (small light grey circles, highlighed by horizontal
arrows) were bound to the SK-BR-3 cells (larger dark grey circles,
highlighed by vertical arrows in FIG. 5C, no arrows in FIG. 5D)
after washing compared to cells incubated with the mixture of
unconjugated Fabs (FIG. 5C-D), confirming heterodimer-mediated
cell-cell interaction. FIG. 5C shows the overlay of SK-BR-3 cells
and Jurkat cells treated with the anti-HER2/anti-CD3 Fab
heterodimer. FIG. 5D shows the overlay of SK-BR-3 cells and Jurkat
cells treated with a 1:1 mixture of unconjugated anti-CD3 and
anti-HER2 Fabs.
Example 5
In Vitro Cytotoxicity of Anti-Her2/Anti-CD3 Heterodimer
[0241] Next, we demonstrated that the heterodimer could recruit T
cells to kill the target cancer cells in an in vitro cytotoxicity
assay. Human PBMCs (peripheral blood mononuclear cells) were
purified with Ficoll (GE Healthcare) from fresh blood of healthy
donors and mixed with target cells, HER2-transfected MDA-MB-435
(33), at an effector to target cell ratio of 10 to 1
(1.times.10.sup.5 to 1.times.10.sup.4 cells) in RPMI media
supplemented with 10% FBS. Non-transfected MDA-MB-435 cells were
used as an isogenic negative control. A 1:1 mixture of unconjugated
anti-HER2 and anti-CD3 Fabs were used as an additional negative
control to further demonstrate that cytotoxicity was due to
bispecific antibody-based interactions. After incubation for 16
hours at 37.degree. C., the amount of LDH (lactate dehydrogenase)
was measured from lysed cells as an indicator of cytotoxicity (9).
FIG. 6 shows the dose-dependent cytotoxicity with MDA-MB-435/HER2+
cells in the presence of human PBMCs and antibody heterodimer.
Different concentrations of anti-HER2/anti-CD3 Fab heterodimer
(circles, line 1), or a 1:1 mixture of unconjugated anti-HER2 Fab
and anti-CD3 Fab (square, line 2) were incubated with
MDA-MB-435/HER2+ cells. Heterodimer (upward triangle, line 3) or a
mixture (downward triangle, line 4) of anti-HER2 Fab and anti-CD3
Fab were incubated with MDA-MB-435/HER2- cells as negative
controls. After 17 hours of incubation at 37.degree. C. and 5%
CO.sub.2, cytotoxicity was measured by levels of LDH (lactate
dehydrogenase) release from lysed cells using the Cytotox 96
Nonradioactive Cytotoxicity Assay Kit (Promega). In separate wells,
MDA-MB-435/HER2+ or MDA-MB-435/HER2- cells with no PBMCs were
incubated and lysed using the lysis buffer (provided in the assay
kit) as maximum cytotoxicity controls. The absorbance at 490 nm was
recorded using SpectraMax 250 plate reader (Molecular Devices
Corp.). Percent cytotoxicity was calculated by:
% Cytotoxicity = ( Absorbance expt - Absorbance spontaneous average
) ( Absorbance ma x average - Absorbance spontaneous average )
##EQU00001##
[0242] As shown in FIG. 6, lysis of HER2 positive target cells was
observed in a dose dependent manner only when the conjugated
anti-HER2/anti-CD3 heterodimer was present (FIG. 6, cicles, line
1); the half maximal effective concentration (EC.sub.50) was
.about.20 pM. HER2-negative MDA-MB-435 cells were not affected by
the heterodimer, and neither cell line was affected by the
unconjugated Fab mixture. The percent cytoxicity of the HER2
positive and HER2 negative cells is shown in Table 1.
TABLE-US-00001 TABLE 1 Percent Cytotoxicity of HER2 positive or
HER2 negative cells treated with an anti-HER2/anti-CD3 Fab
heterodimer or a 1:1 mixture of unconjugated anti-HER2 Fab and
anti-CD3 Fab. % cycotoxicitiy pM HER2+/conj Her2+/unconj Her2-/conj
Her2-/unconj 0.0000 -2.1252 -0.1635 5.2564 1.2786 0.0381 1.1444
-0.6539 4.9723 -0.1421 0.1526 0.1635 -0.3270 7.2453 1.5627 0.6104
3.1061 -1.1444 2.6992 1.8469 2.4414 11.7705 4.2505 10.9391 6.1088
9.7656 21.7427 4.7409 9.2343 4.4040 39.0625 66.5359 5.2313 11.7915
6.9612 156.2500 93.1829 4.9044 12.6438 6.3930 625.0000 93.3464
4.5774 16.0534 1.8469
[0243] Additionally, cell lysis and formation of aggregates could
be directly observed under a microscope in the heterodimer-present
HER2 positive cell sample, but not in any of the controls (FIG. 7).
FIG. 7A shows the microscopic images from the cytoxicity assay in
HER2- cells treated with the 1:1 mixture of unconjugated anti-HER2
Fab and anti-CD3 Fab. FIG. 7B shows the microscopic images from the
cytoxicity assay in HER2- cells treated with the anti-HER2/anti-CD3
Fab heterodimer. FIG. 7C shows the microscopic images from the
cytoxicity assay in HER2+ cells treated with the 1:1 mixture of
unconjugated anti-HER2 Fab and anti-CD3 Fab. FIG. 7D shows the
microscopic images from the cytoxicity assay in HER2+ cells treated
with the anti-HER2/anti-CD3 Fab heterodimer.
Example 6
In Vivo Cytotoxicity of Anti-Her2/Anti-CD3 Heterodimer
[0244] Since both anti-Her2 and anti-CD3 Fabs recognize human
epitopes, human cancer cells and human T cells must be introduced
into immuno-deficient NOD-SCID (NOD.CB17-Prkdc.sup.scid/NCrCrl)
mice. Others have also reported better efficiency with NOD-SCID
IL2-gamma-knockout mice (34) or by supplementing with irradiation
(35). The simplest model includes premixing and co-injecting the
tumor cells and purified human PBMCs (36, 37). While, this works
for a preventative treatment model, it is not ideal for treatment
models. Tumor cells, like MDA-MB-435 Her2-transfected, often take
1-2 weeks to reach the 100 mm.sup.3 tumor size that is often
required for treatment models before administrating drug. A
complication is that hPBMCs have been reported to only remain in
NOD-SCID mice for 2 weeks, potentially reducing the efficacy by the
time drug administration begins (37). Others have explored
intraperitoneal (IP) and intravenous (IV) injection of PBMCs to
reconstitute a human immune system in the mouse (38, 39). This
method is more difficult and often is only retroactively confirmed
of correct reconstitution by performing histology on the spleen.
For the anti-Her2/anti-CD3 Fab heterodimer, we began with the
simplest prevention model.
[0245] First, the best ratio of cancer and non-activated hPBMCs for
premixing was determined. Nine female NOD-SCID mice were divided
into three groups: 1:1 ratio (2.times.10.sup.6
MDA-MB-435/Her2.sup.+ cells, 2.times.10.sup.6 hPBMCs); 1:2 ratio
(2.times.10.sup.6 MDA-MB-435/Her2.sup.+ cells, 4.times.10.sup.6
hPBMCs); and 1:5 (2.times.10.sup.6 MDA-MB-435/Her2.sup.+ cells,
1.times.10.sup.7 hPBMCs). All groups were inject with 1:1 ratio of
premixed cells to MatriGel above the right shoulder by subcutaneous
injection (SC). Tumor size were monitored by caliper measurements
of length and width every 3-4 days and approximated by the
formula:
Tumor volume (mm.sup.3)=(Length.times.width.times.width)/2
[0246] The results showed that the tumors in the 1:1 ratio group
grew extremely fast, tumors in the 1:2 ratio group began to grow
around day 15, and the tumors in the 1:5 ratio group were
suppressed (FIG. 8). FIG. 8 shows growth curves of MDA-MB-435/Her2'
premixed with hPBMC and Matrigel. The ratios of cancer cells to
hPBMCs were 1:1 (square), 1:2 (triangle), and 1:5 (x) with 3
NOD-SCID mice per group. Tumors in 1:1 group grew very fast, tumors
in 1:2 group began to grow after 2 weeks, and tumors in 1:5 group
did not grow at all. As predicted, having too many hPBMCs inhibits
tumor growth. While 1:1 ratio of cancer cells to hPBMCs grew very
rapidly, it would not be ideal as the tumor cells were likely to
overtake the number of hPBMCs very rapidly before the T cells are
able to sufficiently kill the tumor cells. Hence, a 1:2 ratio for
this particular cancer cell line was used for premixed preventative
studies.
[0247] Preventative in vivo xenograft model was conducted by
co-injecting 2.times.10.sup.6 MDA-MB-435/Her2.sup.+ tumor cells,
4.times.10.sup.6 nonactivated human PBMCs, and MatriGel by SC into
female NOD-SCID mice. After 4 days, the mice were grouped for
treatment with anti-Her2/anti-CD3 Fab heterodimer (n=7),
unconjugated anti-Her2 Fab and anti-CD3 Fab (n=7), or PBS (n=6).
Because the serum half-life of the heterodimer was found to be
about 6-7 hours in rats (collaboration with Ambrx, Inc.), six daily
doses were given at 1 mg/kg per dose IV. After 17 days, the group
treated with heterodimer had significant tumor reduction (P<0.05
by one-way ANOVA) compared to unconjugated Fabs and PBS control
(FIG. 9). FIG. 9 shows the results for the preventative in vivo
xenograft model with premixed MDA-MB-435/Her2' cancer cells
(2.times.10.sup.6 cells), hPBMCs (4.times.10.sup.6 cells), and
MatriGel. Treatment of anti-Her2/anti-CD3 Fab heterodimer (circle),
unconjugated anti-Her2 and anti-CD3 (square=Fab mixture), or PBS
(triangle) was given daily by IV starting on Day 4. Table 2 shows
the average tumor volume for conjugate (anti-Her2/anti-CD3 Fab
heterodimer), unconjugate (anti-Her2 and anti-CD3=Fab mixture), or
PBS-treated mice.
TABLE-US-00002 TABLE 2 Average tumor volume Average tumor volume (N
= 7, mm.sup.3) Day Conjugate Unconjugated PBS 2 90.3553 153.0106
111.1696 6 126.2919 274.4486 211.9636 9 71.5163 486.3442 368.4529
13 52.7436 554.7574 442.6591 18 32.3901 714.0962 593.8855 21
35.6919 1383.2782 1025.3079 24 30.4773 1595.4776 30 4.0229 34
7.6792 37 0 42 0 50 0 56 0 65 0 71 0
[0248] While it is likely that there are very few tumor cells left
in the anti-Her2/anti-CD3 Fab heterodimer group, the size did not
reduce to zero. This is likely due to residual MatriGel, which
often stays in the mice for 3-6 weeks after initial injection.
Additionally, a small signal was still observed by IVIS imaging,
but did show a drastic difference in size between heterodimer
treatment and unconjugated groups (FIG. 10). FIG. 10 shows the IVIS
imaging of two heterodimer treatment mice (left two) and two
unconjugated Fabs mice (right two). Luciferin was inject into each
mice and incubated for 10 minutes before capturing the image. The
MDA-MB-435/Her2' were transfected to include the firefly luciferase
gene. Luminescent units are relative. As shown in FIG. 10, ROI
1=6.048e+08 (mouse #1), ROI 2=1.805e+09 (mouse #2), ROI 3=7.751e+09
(mouse #3), and ROI 4=6.380e+09 (mouse #4).
[0249] The MatriGel may have formed a matrix where it protected a
small amount of tumor cells from being killed or small population
of Her2.sup.- or Her2 low cells in the cancer cell line had escaped
death. The excised tumor at the end of Day 20 did confirm that the
majority of the remaining bump in the treatment group was due to
MatriGel, which is often soft and white while tumor is solid and
red (due to blood vessels) (FIG. 11). FIG. 11 shows the excised
tumor or MatriGel from the preventative anti-Her2/anti-CD3 Fab
heterodimer study. The top row is the heterodimer treatment group
(n=7), middle row is unconjugated Fabs group (n=7), and bottom row
is the PBS group (n=6). The "tumors" left from the heterodimer
treatment group is most likely residual MatriGel, which is
characterized by being soft and white.
Example 7
Synthesis of Bifunctional Linkers
[0250] All chemicals were obtained from commercial sources and used
without further purification. 1H- and 13C-NMR spectra were obtained
on a Varian NOVA-399 (400 MHz) or MER-300 (300 MHz)
spectrophotometer. Chemical shift values were recorded as parts per
million relative to tetramethylsilane as an internal standard.
Protein mass spectra were acquired at the Scripps Center for Mass
Spectrometry (La Jolla, Calif.).
[0251] FIG. 12 shows a general scheme for synthesizing bifunctional
linkers. As shown in FIG. 12, Triphenylphosphine (430 mg, 1.64
mmol), N-hydroxyphthalimide (270 mg, 1.66 mmol) and DIAD (0.32 mL,
1.6 mmol) were added to a solution of
1-azido-3,6,9-trioxaundecane-1-ol (300 mg, 1.37 mmol) in
CH.sub.2Cl.sub.2 (20 mL) at 0.degree. C. After stirring overnight
at room temperature, the solvent was removed under reduced
pressure, and the residue was purified by flash column
chromatography on silica gel (Hex-EtOAc, 1:1) to afford compound 5
(426 mg, 85%). R.sub.f 0.2 (Hex-EtOAc, 1:1). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 7.82 (dd, J=5.4, 3.1 Hz, 2H), 7.74 (dd, J=5.5,
3.1 Hz, 2H), 4.38-4.34 (m, 2H), 3.88-3.82 (m, 2H), 3.69-3.62 (m,
4H), 3.62-3.54 (m, 6H), 3.36 (t, J=5.6 Hz, 2H). .sup.13C NMR (100
MHz, CDCl.sub.3): 163.96, 134.98, 129.53, 124.02, 77.76, 71.34,
71.17, 71.17, 71.12, 70.55, 69.83, 51.23. MS (ESI) calcd. for
C16H20N4O6 (M.sup.++Na) 387.1, found 387.1. The structure of
compound 5 is shown as Formula V below.
##STR00001##
[0252] As shown in FIG. 12,
2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)isoindoline-1,3-dione
(compound 5, 426 mg, 1.17 mmol) was dissolved in methanolic ammonia
solution (7 N in MeOH, 50 mL) and stirred overnight at 40.degree.
C. After the solvent was removed under reduced pressure, the crude
mixture was dissolved in CH.sub.2Cl.sub.2 (50 mL), and the white
precipitate was filtered-off. After concentration, the residue was
purified by flash column chromatography on silica gel
(CH.sub.2Cl.sub.2-MeOH, 50:1) to afford compound 2 (254 mg, 93%).
R.sub.f 0.3 (CH.sub.2Cl.sub.2-MeOH, 20:1). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 3.90-3.82 (m, 2H), 3.70-3.67 (m, 4H), 3.66 (s,
6H), 3.67-3.65 (m, 2H), 3.39 (t, J=5.6 Hz, 2H). .sup.13C NMR (100
MHz, CDCl.sub.3): .delta. 74.70, 70.69, 70.62, 70.60, 70.52, 70.01,
69.66, 50.72. HRMS (ESI) calcd. for C.sub.8H.sub.18N.sub.4O.sub.4
(M.sup.++1) 235.1401, found 235.1403. The structure of compound 2
is shown as Formula VI below.
##STR00002##
[0253] As shown in FIG. 12, TEA (0.18 mL, 1.3 mmol) was added to a
solution of
(1R,8S,9R)-bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate
(compound 6, 270 mg, 0.86 mmol) in DMF (10 mL).
O,O'-[oxybis(2,1-ethanediyloxy-2,1-ethanediyl)]bis(hydroxylamine)
(7, 770 mg, 3.4 mmol) in CH.sub.2Cl.sub.2 (40 mL) was added to the
solution and stirred overnight at room temperature. The solvent was
removed by blowing nitrogen gas, and the residue was purified by
flash column chromatography on silica gel (CH.sub.2Cl.sub.2-MeOH,
20:1) to afford 3 (280 mg, 82%). R.sub.f 0.2
(CH.sub.2Cl.sub.2-MeOH, 10:1). .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 8.16 (s, 1H), 5.58 (br. s, 2H), 4.10-4.00 (m, 4H),
3.89-3.81 (m, 2H), 3.78-3.70 (m, 4H), 3.68 (s, 8H), 2.40 (d, J=13.6
Hz, 2H), 2.27 (d, J=13.7 Hz, 2H), 2.16 (d, J=16.1 Hz, 2H),
1.50-1.27 (m, 2H), 0.89-0.62 (m, 3H). .sup.13C NMR (75 MHz,
CDCl.sub.3): .delta. 157.95, 98.98, 75.64, 74.99, 70.81, 70.73,
70.25, 69.84, 69.49, 33.46, 23.80, 23.24, 21.59. HRMS (ESI) calcd.
for C.sub.19H.sub.32N.sub.2O.sub.7 (M.sup.++1) 401.2282, found
401.2287. The structure of compound 3 is shown as Formula VII
below.
##STR00003##
Example 8
Expression of Antibody Fragments in E. coli
[0254] Herceptin-Fab and UCHT1-Fab sequences were obtained from the
literature and inserted into a pBAD vector behind the stII signal
sequence. The sites for unnatural amino acid incorporation
(LC-Ser202 for Her2 Fab, HC-Lys138 for UCHT1) were quickchanged
(Stratagene) to TAG amber nonsense codon. The pBAD vector was
co-transformed with pEVOL-pAcF (a vector containing orthogonal M.
jannaschii tRNA and aminoacyl-tRNA synthetase specific for pAcF in
DH10B cells. The cells were grown in LB media (2 L) supplemented
with 100 mg/ml ampicillin, 25 mg/ml chloramphenicol, and 1 mM pAcF
at 37.degree. C. and 250 rpm. At OD.sub.600 0.8, cells were induced
with 0.2% arabinose and moved to 30.degree. C. for 16 hours at 270
rpm. Cells were harvested and proteins were extracted by incubating
with 150 mL of periplasmic lysis buffer (20% sucrose, 30 mM Tris,
pH 8, 1 mM EDTA, and 0.2 mg/mL lysosyme) for 30 min at 37.degree.
C. Extracts were clarified by centrifugation (18000 rpm, 30 min),
filtered through 0.22 micron filter, and loaded onto Protein G
column (GE healthcare). The column was washed with 20 bed volumes
of 50 mM NaOAc, pH 5.2, and proteins were eluted with 10 bed
volumes of 100 mM glycine, pH 2.8. The eluents were immediately
neutralized by adding 10% of 1 M Tris, pH 8, and dialysed against
PBS for long-term storage.
Example 9
Linker Conjugation and Protein Coupling Reaction
[0255] The antibodies (anti-HER2 Fab, anti-CD3 Fab) were buffer
exchanged into 100 mM NaOAc, pH 4.5, and the concentrations were
adjusted to .about.5 mg/mL. The oxime ligation was conducted with
50 times molar excess of linkers, and the reaction was complete
within 24 hours, as monitored by LC-MS. Excess linkers were removed
by size filtration (Amicon 10K) or size exclusion column (Superdex
75) chromatography, the antibodies were buffer exchanged into PBS,
pH 7.4, and the protein concentrations were adjusted to 10 mg/mL.
Fab-Az conjugate and Fab-cyclooctyne conjugate were mixed at 1:1
molar ratio (.about.10 mg in total), and incubated for 2 days at
37.degree. C. Dimeric Fab conjugate was purified from unreacted
Fabs by size exclusion column (Superdex 200) (FIG. 4B).
Example 10
Flow Cytometry Analysis
[0256] SK-BR-3 cells were trypsinized (0.05% trypsin/EDTA, Hyclone)
and washed with PBS. Jurkat cells in suspension were directly used
without trypsinization. Cells (0.2.times.10.sup.6) were blocked
with 3% BSA in PBS (200 ml) for 1 hour at 4.degree. C. then
incubated with 100 nM of primary antibodies for 1 hour at 4.degree.
C. Cells were washed (cold PBS 1 ml) twice, resuspended in 200 ml
of cold PBS, and incubated with secondary goat anti-human kappa PE
conjugated antibody (100X, Southern Biotech) for 30 min at
4.degree. C. Cells were washed twice with 1 mL of cold PBS before
analysis on a BD LSR II flow cytometer (Beckton Dickinson
Immunocytometry Systems) with 10,000 cell events per sample. Data
was analyzed using FlowJo software (Tree Star Inc.).
Example 11
Fluorescent Staining
[0257] SK-BR-3 and Jurkat cells were stained with Mito Trakcer Red
(Invitrogen) and CFSE (carboxyfluorescein succinimidyl ester,
Invitrogen), respectively, following the manufacturer's protocol.
Jurkat cells (2.times.10.sup.4) were incubated with
anti-HER2/anti-CD3 heterodimer (100 nM) in 200 mL of PBS for 30 min
at 4.degree. C. In a separate tube, Jurkat cells were incubated
with a 1:1 mixture of anti-HER2 Fab and anti-CD3 Fab (100 nM each)
as negative controls. After washing with 1 mL of cold PBS, the
Jurkat cells were resuspended with 200 ml of RPMI media with 10%
FBS, then mixed with SK-BR-3 cells (2.times.10.sup.3) in the same
media (200 mL). Two hundred microliters of the cell mixture was
added into a clear bottom 96 well plate, and incubated at
37.degree. C. and 5% CO.sub.2. After 6 hours, wells were gently
washed with PBS (200 mL) 4 times and imaged on a fluorescent
microscope (Eclipse Ti, Nikon) under FITC (for CFSE) and rhodamine
(for Mitro Traker Red) filters. The images from each filter were
combined to produce an overlay image as seen in FIG. 7C.
Example 12
In Vitro Cytotoxicity Assays
[0258] Peripheral blood mononuclear cells (PBMCs) were purified
from fresh healthy human donor blood by conventional Ficoll-Hypaque
gradient centrifugation. Purified PBMCs were washed and incubated
in flasks in RPMI media with 10% FBS for 2 hours to remove any
adherent cells, and then transferred to anti-CD3 (eBioScience) and
anti-CD28 (eBioScience) antibody-coated ELISA plates at 37.degree.
C. After 3 days, the PBMCs were transferred into a flask and
incubated with 20 units/mL IL2 (R&D Systems) for T cell
proliferation. HER2-transfected MDA-MB-435 or non-transfected
MDA-MB-435 cells (target cells) were dissociated with 0.05%
tryspin/EDTA solution (HyClone) and washed with RPMI with 10% FBS.
1.times.10.sup.4 target cells were mixed with PBMCs at 1:10 ratio
in 100 .mu.l, and incubated with different concentrations of
conjugated and unconjugated anti-Her2/anti-CD3 Fabs (10 .mu.L in
PBS) for 17 hours at 37.degree. C. Cytotoxicity of each well was
measured for LDH (lactate dehydrogenase) levels in supernatant
using Cytotox-96 non-radioactive cytotoxicity assay kit (Promega).
Lysis solution (10 .mu.L, provided in the same kit) was added to
wells with only target cells to get the maximum killing, and
spontaneous killing was measured from wells with effector and
target cells treated with vehicle (10 .mu.L PBS). The absorbance at
490 nm was recorded using SpectraMax 250 plate reader (Molecular
Devices Corp.). Percent cytotoxicity was calculated by:
% Cytotoxicity = ( Absorbance expt - Absorbance spontaneous average
) ( Absorbance ma x killing average - Absorbance spontaneous
average ) ##EQU00002##
Example 13
In Vivo Xenograft Models
[0259] Preventative in vivo xenograft model of bispecific
antibodies was conducted by implanting pre-mixed
MDA-MB-435/Her2.sup.+ cells (2.times.10.sup.6), non-activated PBMC
(4.times.10.sup.6), and MatriGel on the right shoulder of female
NOD-SCID (NOD.CB17-Prkdc.sup.scid/NCrCrl) mice by SQ. After 2-4
days, mice were randomized and grouped into 6-7 mice per group and
given compound by IV. For the BiFab, conjugate, unconjugate, or PBS
was given daily at 1 mg/kg for 6-10 days. Tumors were monitored
twice a week by caliper measurements of length, width, and height
and the tumor volume was estimated by: Tumor
volume=(length.times.width.times.height)/2.
[0260] Weight was also monitored. All procedures were approved by
The Scripps Research Institute Animal Care and Use Committee and
were performed according to national and institutional guidelines
for the humane treatment of animals.
Example 14
Construction of IgG-Based Bispecific Antibodies
[0261] The first step of bispecific antibody construction using our
method was conjugating the bifunctional linker to the respective
antibody components. Linker bearing the azide was conjugated to
anti-Her2 IgG and Fab, while the linker bearing cycloocyne was
conjugated to anti-CD3 Fab single (HC-K138X) and double mutant
(LC-5202X, HC-K138X). The oxime ligation was conducted with 50-fold
molar excess of linker in acetate buffer pH 4.5 at 37.degree. C.
for 16-48 hours. Complete conjugation was monitored by LCMS of the
anti-CD3 Fab conjugation reaction (IgG reaction would have required
additional preparation for LCMS). Excess linker was removed by size
exclusion chromatography or excess washing in 30 kDa or 10 kDa
Amicon concentration columns (Millipore). Antibody-linkers were
buffer exchanged into PBS for the second step of conjugation. For
Durga, anti-Her2-IgG-Az.sub.2 and anti-CD3-Fab-Oct (single mutant)
were incubated at 1:8 molar ratio together and concentrated to 10
mg/mL total. For Fiddler, anti-Her2-IgG-Az.sub.2 was
anti-CD3-Fab-Oct.sub.2 (double mutant) were incubated at 1:1 molar
ratio at a final concentration of only 1 mg/mL to encourage
intramolecular reaction over intermolecular reaction. The Podracer
compound utilized anti-Her2-Fab-Az and anti-CD3-Fab-Oct (single
mutant) at 1:8 molar ratio at a final concentration of 10 mg/mL.
All constructs were incubated at 37.degree. C. and monitored every
other day by SDS-PAGE for reaction completion, which usually took
about 7-10 days. All conjugates were purified by Superdex 200 size
exclusion column at 0.2 mL/min with PBS+100 mM NaCl as the mobile
phase. SDS-PAGE was used to analyze each 0.5 mL fraction and the
most pure fractions were combined and used for subsequent testing.
Purified conjugates were then further analyzed by non-reducing and
reducing SDS-PAGE gels.
Example 15
Western Blot of Her2 Receptor Expression Levels
[0262] The Her2 expression level on different cell lines were
determined by Western blot. 1.times.10.sup.4 Her2 positive cells
(SK-BR-3, HCC-1954, MDA-MB-435/Her2.sup.+) and 1.times.10.sup.6
Her2 "negative" cells (MDA-MB-231, MDA-MB-435/Her2.sup.-, CHO) were
washed once with PBS and resuspended in 30 .mu.L of Cell Lytic M
buffer (Sigma) and incubated at 4.degree. C. for 30 min. The
solution was centrifuged and the supernatant was mixed with SDS
loading buffer for SDS-PAGE. Semi-dry transfer to PVDF membrane
(Invitrogen) was conducted, and the membrane was blocked with 3%
milk (Biorad) in PBS with 0.01% Tween (blocking buffer) for 1 hr at
room temperature. Primary anti-Her2-IgG antibody (the same on used
for conjugation) was incubated at 1 .mu.g/mL in blocking buffer at
4.degree. C. overnight. The membrane was washed 4 times with
PBS+0.01% Tween for 10 min each. Anti-kappa-HRP secondary (Sigma)
was then incubated at room temperature for an hour and washed 4
more times with PBS+0.01% Tween. SuperSignal West Durga Extended
Duration Substrate (Thermo Scientific) was incubated with the
membrane for 10 minutes, washed once with PBS+0.01% Tween, and
developed on a SRX-101A developer (Konica Minolta) with Hyblot CL
film (Denville Scientific).
Example 16
Expression of Proteins with Unnatural Amino Acids in E. coli
[0263] The general method for expressing proteins such as Z-domain,
T4 lysozyme (T4L) and LamB transmembrane protein were similar to
that which was previously described (2). The gene of interest (with
TAG in the codon position that is desired to be modified with the
UAA) is usually on one plasmid and the desired tRNA and amino-acyl
tRNA synthetase pair are on a different plasmid with a different
selection marker and origin of replication. The most optimized
system to date is the pEVOL plasmid, which has one optimized
tyrosyl-tRNA that responds to the amber codon and two copies of RS:
one behind a constitutive promoter to provide a basal level of
expression and one behind the arabinose promoter for strong
inducible expression. The pEVOL plasmid (chloramphenicol
resistance) is also very compatible with pET vector (ampicilin
resistance), which is a common vector for protein expression but is
restricted to BL21(DE3) cells. The co-transformed cells were grown
in 2.times.YT or LB (required for LamB expression) at 37.degree. C.
overnight or until saturation. The cultures were then inoculated at
OD.sub.600 of 0.2 into fresh media containing 1 mM UAA at
37.degree. C. At OD.sub.600 of 0.8-1.0, 1 mM IPTG and 0.02%
arabinose were added and the cultures were moved to 30.degree. C.
for overnight growth. The next day, cells were harvested for
downstream application. Z-domain and T4L that contain C-terminal
His6 tags were sonicated, clarified of precipitates, and incubated
with Ni-NTA resin at room temperature for 30 min. Resin was washed
twice in desktop centrifuge columns and eluted with buffer
containing immidozole. For conjugation, protein was often buffer
exchanged into PBS using Amicon centrifugal columns.
Example 17
Analysis of Anti-CD20/Anti-CD3 Bispecific Antibody
[0264] Anti-CD20 (for B cell lymphoma) and anti-CD3 Fab fragments
and heterodimers were constructed according to methods as described
in the previous examples. Briefly, we incorporated pAcF into
anti-CD20 Fabs (S202pAcF), modified them with bifunctional linkers,
and conjugated to anti-CD3 Fab to prepare anti-CD20/anti-CD3
bispecific antibody. All of the conjugation and purification steps
followed the procedure described for the preparation of the
anti-Her2/anti-CD3 bispecific antibody.
[0265] We also carried out in vitro cytotoxicity assays where we
mixed target cells (e.g., Ramos cells) with hPBMCs at 1:10 ratio,
and incubated with different concentrations of bispecific
antibodies. Cytotoxicity was determined by detecting LDH from lysed
cells as described in Example 17. FIG. 13 depicts a graph of the
cytoxicity of Ramos cells treated with an anti-CD20/anti-CD3
bispecific antibody (circle) or treated with unconjugated Fab
fragments (square). Table 3 shows the cytoxicity of Ramos cells
treated with an anti-CD20/anti-CD3 bispecific antibody or treated
with unconjugated Fab fragments.
TABLE-US-00003 TABLE 3 Cytotoxicity of Ramos cells Relative
Fluorescence Units (RFU) pM aCD20/aCD3 conjugated unconjugated 0
0.689 0.7075 0.038147 0.6775 0.6895 0.1525879 0.6935 0.7235
0.6103516 0.7175 0.723 2.441406 0.7075 0.7415 9.765625 0.7195
0.7205 39.0625 0.7535 0.685 156.25 0.8345 0.687 625 0.967 0.676
2500 1.029 0.663 10000 1.03 0.6655
Example 18
Analysis of Anti-EGFR/Anti-CD3 Bispecific Antibody
[0266] Anti-EGFR (for KRAS/BRFA mutated colorectal or lung cancer)
and anti-CD3 Fab fragments and heterodimers were constructed
according to methods as described in the previous examples.
Briefly, we incorporated pAcF into anti-EGFR Fabs (S202pAcF),
modified them with bifunctional linkers, and conjugated to anti-CD3
Fab to prepare anti-EGFR/anti-CD3 bispecific antibody. All of the
conjugation and purification steps followed the procedure described
for the preparation of the anti-Her2/anti-CD3 bispecific
antibody.
[0267] We also carried out in vitro cytotoxicity assays where we
mixed target cells (AGS, A549, or HT29 cells) with hPBMCs at 1:10
ratio, and incubated with different concentrations of bispecific
antibodies. Cytotoxicity was determined by detecting LDH from lysed
cells as described in Example 17. FIG. 14 and Table 4 shows the
cytoxicity of A549 cells treated with an anti-EGFR/anti-CD3
bispecific antibody (circle) or treated with unconjugated Fab
fragments (square). FIG. 15 and Table 5 shows the cytoxicity of
HT29 cells treated with an anti-EGFR/anti-CD3 bispecific antibody
(circle) or treated with unconjugated Fab fragments (square). FIG.
16 and Table 6 shows the cytoxicity of AGS cells treated with an
anti-EGFR/anti-CD3 bispecific antibody (circle) or treated with
unconjugated Fab fragments (square).
TABLE-US-00004 TABLE 4 Cytoxicity of A549 cells Relative
Fluorescence Units (RFU) pM aEGFR/aCD3 conjugated unconjugate
0.6104 1.3377 0.9040 2.4414 1.3780 0.8793 9.7656 1.3857 1.0233
39.0625 1.5187 0.9810 156.25 1.9837 0.9683 625 2.2673 0.9590 2500
2.3063 1.0847 10000 2.2890 1.2483
TABLE-US-00005 TABLE 5 Cytotoxicity of HT29 cells Relative
Fluorescence Units (RFU) pM aEGFR/aCD3 conjugated unconjugate
0.6104 0.2390 0.2333 2.4414 0.2447 0.2410 9.7656 0.2430 0.2423
39.0625 0.2363 0.2363 156.25 0.2500 0.2390 625 0.3923 0.2407 2500
0.5000 0.2313 10000 0.5380 0.2453
TABLE-US-00006 TABLE 6 Cytoxicity of AGS cells Relative
Fluorescence Units (RFU) pM aEGFR/aCD3 conjugated unconjugate
0.6104 0.2717 0.2500 2.4414 0.2717 0.2560 9.7656 0.2760 0.2567
39.0625 0.2763 0.2657 156.25 0.3003 0.2630 625 0.3663 0.2670 2500
0.4147 0.2570 10000 0.4430 0.2763
Example 19
Expression and Cytotoxicity of UCHT1-CS1 Bispecific Antibody
[0268] BiFab antibodies were created by expression of the UCHT1 Fab
harboring a p-acetylphenylalanine (pAcF) residue in place of lysine
at residue 138 (K138X). The protein was expressed in E. coli,
purified by affinity column and conjugated to the Tet-TEG-ONH2
heterobifunctional linker via oxime ligation using previously
reported conditions (buffer pH 4.5, 30-fold excess, overnight). The
CS1 antibody was expressed in similar fashion using previously
reported conditions. These antibodies harbored the pAcF residue in
place of the serine 202 residue (S202X). The CS1 Fab was
independently conjugated to TCO-TEG-ONH2 (subsequent generations
were produced using a TCO linker (FIG. 2)). Conjugated CS1 was
ligated to UCHT1-Tet Fab using previously reported "click"
chemistry. Antibodies were purified over size-exclusion
chromatography before testing in cytotoxicity assays.
Expression and Purification:
[0269] Fabs were expressed in E. coli as follows. Starter cultures
of E. coli DH10B containing pBAD plasmid expressing the Fab and
pUltra plasmid expressing the orthogonal tRNA/AARS pair for pAcF
incorporation were grown overnight. Cultures were subsequently
diluted to an OD600=0.05, grown in the presence of 1 mM pAcF at
37.degree. C. with shaking at 250 rpm, and induced at OD600=1.0
with 0.2% arabinose and 1 mM IPTG. After induction, culture
conditions were shifted to 26.degree. C. with shaking at 250 rpm,
and the cells allowed to grow for 20-24 h. Cells were harvested at
5 k rpm for 30 min and cell pellets were frozen at -80.degree. C.
followed by thawing at room temperature. Cell pellets were
resuspended in periplasmic lysis buffer (30 mM Tris-HCl, 1 mM EDTA,
20% sucrose pH 7.5 and 0.2 mg/mL lysozyme) and the cell suspension
incubated at 37.degree. C. with shaking for 20-30 min. Lysates were
cleared at 14,000 rpm for 45 min and filtered through a 0.2 .mu.M
filter to remove particulates.
[0270] The cleared and filtered lysates were applied to
pre-equilibrated columns packed with Protein G Sepharose resin (GE
Healthcare), washed with 50 mM sodium acetate (pH 5.0), and protein
eluted with 100 mM glycine (pH 2.8).
Conjugation:
[0271] Purified Fabs were buffer-exchanged into 100 mM sodium
acetate (pH 4.5) 3 times using Amicon concentrators (10,000 MWCO).
Fab (.about.5 mg/mL) was conjugated to the appropriate
heterobifunctional linker (TCO-TEG-ONH2 for UCHT1, and Tet-TEG-ONH2
for CS1) (FIG. 17B) at 50-fold excess linker to Fab, overnight at
37.degree. C. (oxime ligation). The reactions were buffer exchanged
into PBS pH 7.5 and purified by size-exclusion chromatography
(Superdex 200), and the products confirmed by mass
spectrometry.
[0272] Coupling of CS1 Fab to UCHT1 Fab was carried out by mixing
the two Fab-linkers in a 1:1 molar ratio and incubating at
37.degree. C. for .about.16 h. BiFab was purified using
size-exclusion chromatography (Superdex 200), and the product
confirmed by SDS-PAGE (FIG. 17A) and mass spectrometry
analysis.
In Vitro Cytotoxicity:
LDH Assay
[0273] Peripheral blood mononuclear cells (PBMCs) were purified
from fresh healthy human donor blood (Scripps Clinical Research) by
conventional Ficoll-Hypaque gradient centrifugation. Purified PBMCs
were washed and incubated in flasks in RPMI 1640 medium with 5% FBS
for 1 hour at 37.degree. C., before washing with medium and
adjusting concentration to 2.times.10 6 cells/ml. Target cells
MM1.S (CS1+) were washed with RPMI 1640 medium with 5% FBS and
adjusted to a concentration of 0.2.times.10 6 cells/ml. Equal
volumes of target cells and effectors were mixed together to obtain
an effector:target ratio of 10:1, and dispensed in 100 .mu.L
volumes in a 96-well round-bottom plate. The effector-target
mixtures were incubated with different concentrations of BiFabs and
unconjugated Fabs single or mixed (ranging from 10 nM down to 50
fM) for 24 hours at 37.degree. C. Cytotoxicity of each well was
measured as levels of LDH (lactate dehydrogenase) in supernatant
using Cytotox-96 non-radioactive cytotoxicity assay kit (Promega).
Lysis solution provided in the same kit, was added to wells with
only target cells to get the maximum killing, and spontaneous
killing was measured from wells with untreated effector and target
cells. The absorbance at 490 nm was recorded using EnVision
Multilabel Reader (Perkin Elmer). Percent cytotoxicity was
calculated using the following formula: % Cytotoxicity=(Absorbance
expt-Absorbance spontaneous average)/(Absorbance max killing
average-Absorbance spontaneous average). FIG. 17C shows an
increasing cytotoxicity as a result of increasing the concentration
of the .alpha.CS-1/.alpha.CD3 BiFab.
Mass Spectrometry:
[0274] To analyze the mass of Fab's and conjugates, a 10 ul of
0.1-1.0 mg/mL solution of Fab in PBS buffer was injected onto an LC
Agilent 6520 Q-TOF mass spectrometer equipped with a 150.times.2.1
mm C8 column using a Water/Acetonitrile (0.1% formic acid) gradient
(20%-80% over 10 min). Briefly, electrospray settings were as
follows: gas temp 350 deg C, drying gas 10 l/min, nebulizer 40
psig, fragmentor 200, VCap 4500, and TOF settings were standard
(3200) range, 2 GHz, extended dynamic range. The region of the
chromatogram in which the Fab eluted was deconvoluted using the
Agilent Qualitative Analysis software with the pMod algorithm. FIG.
19 A-C shows the deconvolution results for unconjugated CS1 and
UCHT1, CS1 and UCHT1 conjugated to their respective linkers, and
the CS1.times.UCHT1 biFab, and Table 7 shows the summary of
masses.
TABLE-US-00007 TABLE 7 Summary of CS1/UCHT1 Masses delta Sample
Expected Mass Obtained Mass (obtained - expected) BiFab 96963.77
96959.81 -3.96 CS1xUCTH1 CS1-tet 48329.68 48322.4 -7.28 UCHT1-TCO
48664.09 48665.62 1.53 CS1 47911.47 47902.74 -8.73 UCHT1 48305.88
48308.01 2.13
Example 20
Expression and Cytotoxicity of Anti-CD33/Anti-CD3 and
Anti-CLL-1/Anti-CD3 Bispecific Antibodies
Synthesis
[0275] Anti-CLL1 and anti-CD3 Fab containing unnatural amino acid
(pAcPhe) at heavy chain 138 position were expressed following the
standard protocol. The Fab fragments were modified with
hetero-bifunctional linkers and conjugated with anti-CD3 Fab, also
following standard protocol described in the synthesis of other
bispecific antibodies.
Flow Cytometry Analysis
[0276] HL60 (CD33 and CLL-1 positive) or CD3-positive Jurkat cells
(0.2.times.10.sup.6) were blocked with 3% BSA in PBS (200 ml) for 1
hour at 4.degree. C. then incubated with 100 nM of primary
antibodies for 1 hour at 4.degree. C. Cells were washed (cold PBS 1
mL) twice, resuspended in 0.2 mL of cold PBS, and incubated with
secondary goat anti-human kappa PE conjugated antibody (100X,
Southern Biotech) for 30 min at 4.degree. C. Cells were washed
twice with 1 mL of cold PBS before analysis on a BD LSR II flow
cytometer (Beckton Dickinson Immunocytometry Systems) with 10,000
cell events per sample. To get the FACS-based binding graph (FIG.
20A, anti-CLL-1/anti-CD3, FIG. 20B, anti-CD33/anti-CD3), the above
procedure was repeated in round-bottomed 96-well format with
different concentrations of primary antibodies. Cells were injected
using high-throughput sampler (BD HTS). Data was analyzed using
FlowJo software (Tree Star Inc.).
In Vitro Cytotoxicity Assay:
[0277] Purified PBMCs were washed and incubated in flasks in RPMI
1640 medium with 5% FBS for 1 hour at 37.degree. C., before washing
with medium and adjusting concentration to 2.times.10 6 cells/ml.
HL60 cells were stained using PKH26 Red Fluorescent Cell linker kit
(Sigma Aldrich) and the volume adjusted to give a final
concentration of 0.2.times.10 6 cells/ml. Equal volumes of target
cells and effectors were mixed together to obtain an
effector:target ratio of 10:1, and dispensed in 100 .mu.L volumes
in a 96-well round-bottom plate. The effector-target mixtures were
incubated with different concentrations of BiFabs and unconjugated
Fabs single or mixed for 24 hours at 37.degree. C. After 24 hours,
wells were treated with 7-aminoactinomycin D (7-AAD, BD
Biosciences) for 30 min at room temperature to stain dead cells.
For flow cytometry, unstained target cells, target cells stained
with PKH26 only, and target cells stained with 7-AAD only (after
treatment with ice-cold 90% methanol on ice for 30 min) were used
as controls. Samples were read using LSRII Flow Cytometer (BD), and
analyzed using FlowJo. Percentage viability was calculated as the
ratio of live target cells in treated population to that in the
untreated population (see FIG. 21A anti-CD33/anti-CD3 and FIG. 21B
anti-CLL-1/anti-CD3).
Cytokine Release Assay
[0278] HL60 (CLL1.sup.+CD33.sup.+) or RS4:11 (CLL1.sup.-CD33.sup.-)
cells were mixed with non-activated PBMCs (E:T=10:1), and treated
with BiFabs at 25 nM for 24 hours. IFN-.gamma. and IL-2 in
supernatant were measured by ELISA (DuoSet ELISA Kit, R&D
systems) (see FIG. 22A for IFN-.gamma. and FIG. 22B for IL-2).
TABLE-US-00008 TABLE 8 Mass of UCHT1/anti-CD33 and UCHT1/anti-CLL-1
bispecific antibodies Observed Expected Mass Mass UCHT1/Anti-CD33
pAcF 48440 48432 UCHT1/Anti-CD33-Az linker 48656 48653
UCHT1/Anti-Cll-1 pAcF 48496 48497 UCHT1/Anti-Cll-1-Az linker 48712
48710
Example 21
Expression and Cytotoxicity of Anti-EGFRvIII/Anti-CD3 Bispecific
Antibody
[0279] BiFab antibodies were created by expression of the UCHT1 Fab
harboring at p-acetylphenylalanine (pAcF) residue in place of
lysine at residue 138 (K138X). The protein was expressed in E.
coli, purified by affinity column and conjugated to the
TCO-TEG-ONH2 heterobifunctional linker (FIG. 17B) via oxime
ligation (buffer pH 4.5, 30-fold excess, overnight. Hu806 antibody
was expressed in similar fashion using previously reported
conditions. These antibodies harbored the pAcF residue in place of
the serine 202 residue (S202X). Hu806 S202LC pAcF Fab was
independently conjugated to Tet-TEG-ONH2 (FIG. 17B) Hu806-Tet Fab
was then ligated to UCHT1-TCO Fab using "click" chemistry.
Antibodies were purified over size-exclusion chromatography before
testing in cytotoxicity assays.
Expression and Purification:
[0280] Fabs were expressed in E. coli as follows. Starter cultures
of E. coli DH10B containing pBAD plasmid expressing the Fab and
pUltra plasmid expressing the orthogonal tRNA/AARS pair for pAcF
incorporation were grown overnight. Cultures were subsequently
diluted to an OD600=0.05, grown in the presence of 1 mM pAcF at
37.degree. C. with shaking at 250 rpm, and induced at OD600=1.0
with 0.2% arabinose and 1 mM IPTG. After induction, culture
conditions were shifted to 26.degree. C. with shaking at 250 rpm,
and the cells allowed to grow for 20-24 h. Cells were harvested at
5 k rpm for 30 min and cell pellets were frozen at -80.degree. C.
followed by thawing at room temperature. Cell pellets were
resuspended in periplasmic lysis buffer (30 mM Tris-HCl, 1 mM EDTA,
20% sucrose pH 7.5 and 0.2 mg/mL lysozyme) and the cell suspension
incubated at 37.degree. C. with shaking for 20-30 min. Lysates were
cleared at 14,000 rpm for 45 min and filtered through a 0.2 .mu.M
filter to remove particulates. The cleared and filtered lysates
were applied to pre-equilibrated columns packed with Protein G
Sepharose resin (GE Healthcare), washed with 50 mM sodium acetate
(pH 5.0), and protein eluted with 100 mM glycine (pH 2.8).
Conjugation:
[0281] Purified Fabs were buffer-exchanged into 100 mM sodium
acetate (pH 4.5) 3 times using Amicon concentrators (10,000 MWCO).
Fab (.about.5 mg/mL) was conjugated to the appropriate
heterobifunctional linker (TCO-TEG-ONH2 for UCHT1, and Tet-TEG-ONH2
for hu806) at 50-fold excess linker to Fab, overnight at 37.degree.
C. (oxime ligation). The reactions were buffer exchanged into PBS
pH 7.5 and purified by size-exclusion chromatography (Superdex
200), and the products confirmed by mass spectrometry.
[0282] Coupling of hu806 Fab to UCHT1 Fab was carried out by mixing
the two Fab-linkers in a 1:1 molar ratio and incubating at
37.degree. C. for .about.16 h. BiFab was purified using
size-exclusion chromatography (Superdex 200), and the product
confirmed by SDS-PAGE (FIG. 23) and mass spectrometry analysis
(FIG. 24).
In Vitro Cytotoxicity:
[0283] Peripheral blood mononuclear cells (PBMCs) were purified
from fresh healthy human donor blood (Scripps Clinical Research) by
conventional Ficoll-Hypaque gradient centrifugation. Purified PBMCs
were washed and incubated in flasks in RPMI 1640 medium with 5% FBS
for 2 hour at 37.degree. C., before washing with medium and
transferring to an anti-CD3 (eBioScience) coated flask (5
.mu.g/mL). Anti-CD28 (eBioScience) was then added to the cell
solution (2 .mu.g/mL). After 3 days at 37.degree. C., the PBMCs
were transferred into a new flask and incubated with 20 units/mL
IL2 (R&D Systems) for T cell proliferation.
[0284] Target cells A431 (EGFR+) were washed with RPMI 1640 medium
with 5% FBS and adjusted to a concentration of 0.2.times.10 6
cells/ml. Equal volumes of target cells and effectors were mixed
together to obtain an effector:target ratio of 10:1, and dispensed
in 100 .mu.L volumes in a 96-well round-bottom plate. The
effector-target mixtures were incubated with different
concentrations of BiFabs and unconjugated Fabs single or mixed for
24 hours at 37.degree. C. Cytotoxicity of each well was measured as
levels of LDH (lactate dehydrogenase) in supernatant using
Cytotox-96 non-radioactive cytotoxicity assay kit (Promega). Lysis
solution provided in the same kit, was added to wells with only
target cells to get the maximum killing, and spontaneous killing
was measured from wells with untreated effector and target cells.
The absorbance at 490 nm was recorded using EnVision Multilabel
Reader (Perkin Elmer). Percent cytotoxicity was calculated using
the following formula:
% Cytotoxicity=(Absorbance expt-Absorbance spontaneous
average)/(Absorbance max killing average-Absorbance spontaneous
average) (see FIG. 25)
TABLE-US-00009 TABLE 9 Mass of Hu806-UCHT1 BiFab delta Sample
Expected Mass Obtained Mass (obtained - expected) BiFab 96275.34
96306.16 30.82 Hu806-UCTH1 Hu806- 47625.25 47626.69 1.44 Tetrazine
UCHT1-TCO 48664.09 48665.48 1.39 Hu806 Fab 47206.79 47207.09 0.3
UCHT1 Fab 48305.88 48306.63 0.75
TABLE-US-00010 TABLE 10 Sequences of antibodies or antibody
fragments SEQ ID NO Description SEQUENCE 1 Light chain
ATGAAAAAGAATATCGCATTTCTTCTTGCTAGC of an anti-
ATGTTCGTTTTTTCTATTGCTACAAACGCATAC CD3 clone
GCTGACATCCAGATGACCCAGTCTCCATCCTCC of UCHT1
CTGTCTGCATCTGTAGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGGACATCCGTAATT
ATCTGAACTGGTATCAGCAGAAACCAGGGAAA GCCCCTAAGCTCCTGATCTATTATACCTCCCGC
CTGGAGTCTGGGGTCCCATCAAGGTTCAGTGG CTCTGGATCTGGGACAGATTACACTCTGACCAT
CAGCAGTCTGCAACCTGAAGATTTTGCAACTT ACTACTGTCAACAGGGTAATACTCTGCCGTGG
ACGTTCGGCCAAGGTACCAAGGTGGAGATCAA ACGAACTGTGGCTGCACCATCTGTCTTCATCTT
CCCGCCATCTGATGAGCAGTTGAAATCTGGAA CTGCCTCTGTCGTGTGCCTGCTGAATAACTTCT
ATCCCAGAGAGGCCAAAGTACAGTGGAAGGTG GATAACGCCCTCCAATCGGGTAACTCCCAGGA
GAGTGTCACAGAGCAGGACAGCAAGGACAGC ACCTACAGCCTCAGCAGCACCCTGACGCTGAG
CAAAGCAGACTACGAGAAACACAAAGTCTACG CCTGCGAAGTCACCCATCAGGGCCTGTCCTCG
CCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 2 Heavy chain
ATGAAAAAGAATATCGCATTTCTTCTTGCATCT of an anti-
ATGTTCGTTTTTTCTATTGCTACAAACGCGTAC CD3 clone
GCTGAGGTGCAGCTGGTGGAGTCTGGAGGAGG of UCHT1
CTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTC CTGTGCAGCCTCTGGGTACTCCTTTACCGGCTA
CACTATGAACTGGGTCCGCCAGGCTCCAGGGA AGGGGCTGGAGTGGGTCGCACTGATTAATCCT
TATAAAGGTGTTTCCACCTATAACCAGAAATTC AAGGATCGATTCACCATCTCCGTAGATAAATC
CAAAAACACGGCGTATCTTCAAATGAACAGCC TGAGAGCCGAGGACACGGCCGTGTATTACTGT
GCTAGAAGCGGATACTACGGCGATAGTGACTG GTATTTTGACGTCTGGGGCCAAGGAACCCTGG
TCACCGTCTCCTCAGCCTCCACCAAGGGCCCAT CGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCA
CCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG GTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCG TGCACACCTTCCCGGCTGTCCTACAGTCCTCAG
GACTCTACTCCCTCAGCAGCGTGGTGACTGTGC CCTCTAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAG GTGGACAAGAAAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACA 3 Light chain ATGAAAAAGAATATCGCATTTCTTCTTGCTAGC of
ATGTTCGTTTTTTCTATTGCTACAAACGCATAC Trastuzumab
GCTGACATCCAGATGACCCAGTCTCCATCCTCC (anti-Her2)
CTGTCTGCATCTGTAGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGGATGTGAATACCG
CGGTCGCATGGTATCAGCAGAAACCAGGGAAA GCCCCTAAGCTCCTGATCTATTCTGCATCCTTC
TTGTATAGTGGGGTCCCATCAAGGTTCAGTGG CAGTAGATCTGGGACAGATTTCACTCTCACCAT
CAGCAGTCTGCAACCTGAAGATTTTGCAACTT ACTACTGTCAACAGCATTACACTACCCCTCCGA
CGTTCGGCCAAGGTACCAAGCTTGAGATCAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAAC TGCCTCTGTCGTGTGCCTGCTGAATAACTTCTA
TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGG ATAACGCCCTCCAATCGGGTAACTCCCAGGAG
AGTGTCACAGAGCAGGACAGCAAGGACAGCA CCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGC CTGCGAAGTCACCCATCAGGGCCTGTCCTCGC
CCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 4 Heavy chain
ATGAAAAAGAATATCGCATTTCTTCTTGCATCT of
ATGTTCGTTTTTTCTATTGCTACAAACGCGTAC Trastuzumab
GCTGAGGTGCAGCTGGTGGAGTCTGGAGGAGG (anti-Her2)
CTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTC CTGTGCAGCCTCTGGGTTCAATATTAAGGACA
CTTACATCCACTGGGTCCGCCAGGCTCCAGGG AAGGGGCTGGAGTGGGTCGCACGTATTTATCC
TACCAATGGTTACACACGCTACGCAGACTCCG TGAAGGGCCGATTCACCATCTCCGCAGACACT
TCCAAGAACACGGCGTATCTTCAAATGAACAG CCTGAGAGCCGAGGACACGGCCGTGTATTACT
GTTCGAGATGGGGCGGTGACGGCTTCTATGCC ATGGACTACTGGGGCCAAGGAACCCTGGTCAC
CGTCTCCTCAGCCTCCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC
TGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA AGGACTACTTCCCCGAACCGGTGACGGTGTCG
TGGAACTCAGGCGCCCTGACCAGCGGCGTGCA CACCTTCCCGGCTGTCCTACAGTCCTCAGGACT
CTACTCCCTCAGCAGCGTGGTGACTGTGCCCTC TAGCAGCTTGGGCACCCAGACCTACATCTGCA
ACGTGAATCACAAGCCCAGCAACACCAAGGTG GACAAGAAAGTTGAGCCCAAATCTTGTGACAA
AACTCACACA 5 Light Chain ATGAAAAAGAATATCGCATTTCTTCTTGCTAGC of
ATGTTCGTTTTTTCTATTGCTACAAACGCATAC Rituximab
GCTCAGATTGTGCTGAGCCAGAGCCCGGCGAT (anti-CD20)
TCTGAGCGCGAGCCCGGGCGAAAAAGTGACCA TGACCTGCCGCGCGAGCAGCAGCGTGAGCTAT
ATTCATTGGTTTCAGCAGAAACCGGGCAGCAG CCCGAAACCGTGGATTTATGCGACCAGCAACC
TGGCGAGCGGCGTGCCGGTGCGCTTTAGCGGC AGCGGCAGCGGCACCAGCTATAGCCTGACCAT
TAGCCGCGTGGAAGCGGAAGATGCGGCGACCT ATTATTGCCAGCAGTGGACCAGCAACCCGCCG
ACCTTTGGCGGCGGCACCAAGCTTGAGATCAA ACGAACTGTGGCTGCACCATCTGTCTTCATCTT
CCCGCCATCTGATGAGCAGTTGAAATCTGGAA CTGCCTCTGTCGTGTGCCTGCTGAATAACTTCT
ATCCCAGAGAGGCCAAAGTACAGTGGAAGGTG GATAACGCCCTCCAATCGGGTAACTCCCAGGA
GAGTGTCACAGAGCAGGACAGCAAGGACAGC ACCTACAGCCTCAGCAGCACCCTGACGCTGAG
CAAAGCAGACTACGAGAAACACAAAGTCTACG CCTGCGAAGTCACCCATCAGGGCCTGTCCTCG
CCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 6 Heavy Chain
ATGAAAAAGAATATCGCATTTCTTCTTGCATCT of
ATGTTCGTTTTTTCTATTGCTACAAACGCGTAC Rituximab
GCTCAGGTGCAGCTGCAGCAGCCGGGCGCGGA (anti-CD20)
ACTGGTGAAACCGGGCGCGAGCGTGAAAATGA GCTGCAAAGCGAGCGGCTATACCTTTACCAGC
TATAACATGCATTGGGTGAAACAGACCCCGGG CCGCGGCCTGGAATGGATTGGCGCGATTTATC
CGGGCAACGGCGATACCAGCTATAACCAGAAA TTTAAAGGCAAAGCGACCCTGACCGCGGATAA
AAGCAGCAGCACCGCGTATATGCAGCTGAGCA GCCTGACCAGCGAAGATAGCGCGGTGTATTAT
TGCGCGCGCAGCACCTATTATGGCGGCGATTG GTATTTTAACGTGTGGGGCGCGGGCACCACCG
TGACCGTGAGCGCGGCGAGCACCAAGGGCCCA TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC
ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCA
GGACTCTACTCCCTCAGCAGCGTGGTGACTGT GCCCTCTAGCAGCTTGGGCACCCAGACCTACA
TCTGCAACGTGAATCACAAGCCCAGCAACACC AAGGTGGACAAGAAAGTTGAGCCCAAATCTTG
TGACAAAACTCACACA 7 Light chain ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCT of
GCTGCTCTGGCTCCCAGGTGCACGATGTGACA Clone C225
TCCTGCTGACCCAGTCCCCCGTGATCCTGTCCG (anti-EGFR)
TGTCCCCTGGCGAGCGGGTGTCCTTCTCCTGCC GGGCCTCCCAGTCCATCGGCACCAACATCCAC
TGGTATCAGCAGCGGACCAACGGCTCCCCTCG GCTGCTGATCAAGTACGCCTCCGAGTCTATCTC
CGGCATCCCTTCCCGGTTCTCCGGCTCCGGCTC TGGCACCGACTTCACCCTGTCCATCAACTCCGT
GGAGTCCGAGGATATCGCCGACTACTACTGCC AGCAGAACAACAACTGGCCTACCACCTTCGGC
GCTGGAACCAAGCTGGAGCTGAAGCGTACGGT GGCTGCACCATCTGTCTTCATCTTCCCGCCATC
TGATGAGCAGTTGAAATCTGGAACTGCCTCTG TTGTGTGCCTGCTGAATAACTTCTATCCCAGAG
AGGCCAAAGTACAGTGGAAGGTGGATAACGCC CTCCAATCGGGTAACTCCCAGGAGAGTGTCAC
AGAGCAGGACAGCAAGGACAGCACCTACAGC CTCAGCAGCACCCTGACGCTGAGCAAAGCAGA
CTACGAGAAACACAAAGTCTACGCCTGCGAAG TCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTGATGA 8 Heavy chain
ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTC of
GCTGTTGCTACGCGTGTCCACTCCCAGGTGCAG Clone C225
CTGAAGCAGTCCGGCCCTGGCCTGGTGCAGCC (anti-EGFR)
TTCCCAGTCCCTGTCCATCACCTGCACCGTGTC CGGCTTCTCCCTGACCAACTACGGCGTGCACTG
GGTGCGCCAGTCCCCCGGCAAGGGCCTGGAGT GGCTGGGCGTGATCTGGTCCGGCGGCAACACC
GACTACAACACCCCTTTCACCTCCCGGCTGTCC ATCAACAAGGACAACTCCAAGTCCCAGGTGTT
CTTCAAGATGAACTCCCTGCAGTCCAACGACA CCGCCATCTACTACTGCGCCAGAGCCCTGACCT
ACTATGACTACGAGTTCGCCTACTGGGGCCAG GGCACCCTGGTGACCGTGTCCGCCGCTAGCAC
CAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCC
TGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGTGGAACTCAGGCGCCCT
GACCAGCGGCGTGCACACCTTCCCGGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCG
TGGTGACCGTGCCCTCCAGCAGCTTGGGCACC CAGACCTACATCTGCAACGTGAATCACAAGCC
CAGCAACACCAAGGTGGACAAGAAAGTTGAG CCCAAATCTTGTGACAAAACTCACACATGCCC
ACCGTGCCCA 9 Light chain DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVA of
wildtype WYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGS anti-CS1
GTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQG antibody TKLEIK 10 Heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYW of wildtype
MSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDK anti-CS1
FIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDG antibody NYWYFDVWGQGTLVTVSS 11
Light chain GAGAACGTGCTCACCCAATCCCCCGCCATTAT of anti-
GTCCGCCTCCCCAGGCGAAAAGGTGACAATGA CLL-1
CCTGCAGGGCCAGCTCCAACGTGATCAGCTCT antibody
TACGTGCACTGGTACCAGCAACGGTCCGGCGC CTCCCCTAAGCTGTGGATCTATAGCACAAGCA
ACCTGGCTTCCGGCGTGCCTGCACGGTTCAGC GGAAGCGGAAGCGGAACAAGTTACTCCCTCAC
CATTTCTAGCGTTGAAGCCGAGGATGCCGCTA CATACTATTGTCAACAGTACAGCGGATACCCC
CTGACCTTCGGAGCCGGCACAAAACTGGAGCT CAAGAGAGCAGCTGCAGCTCCCAGCGTGTTCA
TTTTTCCTCCCTCCGACGAACAACTGAAAAGCG GAACAGCCTCTGTCGTTTGCCTGTTGAACAATT
TCTACCCTAGGGAGGCCAAGGTCCAGTGGAAA GTGGATAACGCTCTGCAAAGCGGAAATTCTCA
GGAAAGCGTTACCGAACAGGATTCTAAGGACT CTACATACTCTCTGTCTAGCACACTCACgctg
agcaaagcagactacgagaaacacaaagtcta cgcctgcgaagtcacccatcagggcctgtcct
cgcccgtcacaaagagcttcaacaggggagagtgt 12 Heavy chain
GACATCCAGCTGCAGGAGAGCGGCCCCGGCCT of anti-
GGTGAAGCCCAGCCAGAGCCTGAGCCTGACCT CLL-1
GCAGCGTGACCGGCTACAGCATCACCAGCGCC antibody
TATTACTGGAACTGGATCCGGCAGTTCCCCGG CAACAAGCTGGAGTGGATGGGCTACATCAGCT
ACGACGGCCGGAACAACTACAACCCAAGCCTG AAGAACCGGATCAGCATCACCCGGGACACCAG
CAAGAACCAGTTTTTCCTGAAGCTGAACAGCG TGACCACAGAGGACACCGCCACCTATTACTGC
GCCAAGGAGGGAGACTACGACGTGGGCAACT ACTACGCCATGGACTACTGGGGCCAGGGCACC
AGCGTGACCGTGTCTAGCGCCCGGACCAAGGG CCCCAGCGTGTTCCCCCTGGCCCCCAGCTCTAA
GAGCACCAGCGGCGGAACCGCCGCTCTGGGCT GCCTGGTGAAGGACTACTTCCCCGAGCCCGTG
ACCGTGAGCTGGAACAGCGGCGCCCTGACCAG CGGCGTGCACACCTTCCCCGCCGTGCTGCAGA
GCTCTGGCCTGTACAGCCTGAGCAGCGTGGTT
ACCGTGCCCAGTTCTTCCCTGGGCACCCAGACC TACATCTGCAACGTGAACCACAAGCCAGCAA
CACCAAGGTGGACAAGAAAGTGGAGCCCAAG AGCTGC 13 Light chain
GATATTCAGATGACCCAGAGCCCGAGCAGCCT of anti-
GAGCGCGAGCGTGGGCGATCGCGTGACCATTA CD33
CCTGCCGCGCGAGCGAAAGCGTGGATAACTAT antibody
GGCATTAGCTTTATGAACTGGTTTCAGCAGAA ACCGGGCAAAGCGCCGAAACTGCTGATTTATG
CGGCGAGCAACCAGGGCAGCGGCGTGCCGAG CCGCTTTAGCGGCAGCGGCAGCGGCACCGATT
TTACCCTGAACATTAGCAGCCTGCAGCCGGAT GATTTTGCGACCTATTATTGCCAGCAGAGCAA
AGAAGTGCCGTGGACCTTTGGCCAGGGCACCA AAGTGGAAATTAAACGAACTGTGGCTGCACCA
TCTGTCTTCATCTTCCCGCCATCTGATGAGCAG TTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG
CTGAATAACTTCTATCCCAGAGAGGCCAAAGT ACAGTGGAAGGTGGATAACGCCCTCCAATCGG
GTAACTCCCAGGAGAGTGTCACAGAGCAGGAC AGCAAGGACAGCACCTACAGCCTCAGCAGCAC
CCTGACGCTGAGCAAAGCAGACTACGAGAAAC ACAAAGTCTACGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAA CAGGGGAGAGTGT 14 Heavy chain
CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGT of GAAAAAACCGGGCAGCAGCGTGAAAGTGAGC
anti-CD33 TGCAAAGCGAGCGGCTATACCTTTACCGATTA antibody
TAACATGCATTGGGTGCGCCAGGCGCCGGGCC AGGGCCTGGAATGGATTGGCTATATTTATCCGT
ATAACGGCGGCACCGGCTATAACCAGAAATTT AAAAGCAAAGCGACCATTACCGCGGATGAAA
GCACCAACACCGCGTATATGGAACTGAGCAGC CTGCGCAGCGAAGATACCGCGGTGTATTATTG
CGCGCGCGGCCGCCCGGCGATGGATTATTGGG GCCAGGGCACCCTGGTGACCGTGAGCAGCGCC
TCCACCAAGGGCCCATCGGTCTTCCCCCTGGCA CCCTCCTCCTAGAGCACCTCTGGGGGCACAGC
GGCCCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAACCGGTGACGGTGTCGTGGAACTCAGGC
GCCCTGACCAGCGGCGTGCACACCTTCCCGGC TGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG GCACCCAGACCTACATCTGCAACGTGAATCAC
AAGCCCAGCAACACCAAGGTCGACAAGAAAG TTGAGCCCAAATCTTGTGGTGGCGGTCACCATC
ACCATCATCACCACCAC 15 Light chain
DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGW of anti-
LQQKPGKSFKGLIYHGTNLDDGVPSRFSGSGSGT EGFRvIII
DYTLTISSLQPEDFATYYCVQYAQFPWTFGGGTK antibody LEIK (Hu806 VL) 16
Heavy chain QLQESGPGLVKPSQTLSLTCTVSGYSISSDFAWN of anti-
WIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITIS EGFRvIII
RDTSKNQFFLKLNSVTAADTATYYCVTAGRGFP antibody YWGQGTLVTVSS (Hu806 HL)
17 Human light KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP chain
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYS constant
LSSTLTLSKADYEKHKVYACEVTHQGLS*SPVTK region SFNRGEC 18 Human heavy
ASTKGPSVFPLAPSSK*STSGGTAALGCLVKDYFP chain
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV constant
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS region (CH1) CDKTHT
[0285] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of the present invention is embodied by the
appended claims.
[0286] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *