U.S. patent application number 17/483630 was filed with the patent office on 2022-03-10 for mixtures of antibodies.
The applicant listed for this patent is Qilu Puget Sound Biotherapeutics Corporation. Invention is credited to Zhi LIU, Martin J. PENTONY, Wei YAN.
Application Number | 20220073615 17/483630 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-10 |
United States Patent
Application |
20220073615 |
Kind Code |
A1 |
YAN; Wei ; et al. |
March 10, 2022 |
MIXTURES OF ANTIBODIES
Abstract
Described herein are antibodies and mixtures of antibodies
optionally produced by a host cell line, nucleic acids encoding the
antibodies and mixtures of antibodies, host cells containing such
nucleic acids, and methods of treatment using the antibodies,
mixtures of antibodies, or nucleic acids encoding the antibodies or
mixtures of antibodies. Also described are methods of producing
mixtures of antibodies in host cells.
Inventors: |
YAN; Wei; (Sammamish,
WA) ; LIU; Zhi; (Shoreline, WA) ; PENTONY;
Martin J.; (Bedford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Qilu Puget Sound Biotherapeutics Corporation |
Bothell |
WA |
US |
|
|
Appl. No.: |
17/483630 |
Filed: |
September 23, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16303611 |
Nov 20, 2018 |
11130808 |
|
|
PCT/US2017/030676 |
May 2, 2017 |
|
|
|
17483630 |
|
|
|
|
62342167 |
May 26, 2016 |
|
|
|
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/32 20060101 C07K016/32; A61P 35/00 20060101
A61P035/00; A61K 9/00 20060101 A61K009/00 |
Claims
1-81. (canceled)
82. A mixture of antibodies comprising two and not more than three
different major antibody species, which comprises (a) a first
antibody comprising two chains of a first heavy chain (HC1), each
having the same first amino acid sequence, and two chains of a
first light chain (LC1), each having the same second amino acid
sequence, and (b) a second antibody comprising two chains of a
second heavy chain (HC2), each having the same third amino acid
sequence, and two chains of a second light chain (LC2), each having
the same fourth amino acid sequence, wherein: (1) the first and
second antibodies are full-length human and/or humanized IgG
antibodies; (2) the HC1 and the HC2 have different amino acid
sequences, and the LC1 and the LC2 have different amino acid
sequences; (3) the first heavy chain constant (CH1) and the light
chain constant (CL) domains of the first antibody and/or the second
antibody comprise one or more partner-directing alterations that
create one or more pairs of contacting cysteine residues, wherein
one cysteine residue of the pair(s) is in the CH1 domain and the
other is in the CL domain of the antibody or antibodies comprising
the pair(s) of contacting cysteine residues, wherein (A) if the
first and/or second antibody comprising the contacting cysteine
residues is (are) (an) IgG1 antibody or antibodies, then the
pair(s) of heavy chain and light chain positions, respectively, of
the pair(s) of contacting cysteine residues in the IgG1 antibody or
antibodies is (are) selected from the group consisting of:
positions 126 and 124; positions 128 and 118; positions 133 and
117; positions 133 and 209; positions 134 and 116; positions 168
and 174; positions 170 and 162; positions 170 and 176; positions
173 and 160; and positions 183 and 176, (B) if the first and/or
second antibody comprising the contacting cysteine residues is
(are) (an) IgG2 antibody or antibodies, then the pair(s) of heavy
chain and light chain positions, respectively, of the pair(s) of
contacting cysteine residues in the IgG2 antibody or antibodies is
(are) selected from the group consisting of: positions 170 and 162;
and positions 173 and 160, and (C) if the first and/or second
antibody comprising the contacting cysteine residues is (are) (an)
IgG4 antibody or antibodies, then the pair(s) of heavy chain and
light chain positions, respectively, of the pair(s) of contacting
cysteine residues in the IgG4 antibody or antibodies is (are)
selected from the group consisting of: positions 126 and 121;
positions 126 and 124; positions 127 and 121; positions 128 and
118; positions 168 and 174; positions 170 and 162; and positions
173 and 162; and (4) if both the first and second antibodies
comprise a pair of contacting cysteine residues as recited in (3)
above, then the first and second antibodies do not comprise
contacting cysteine residues at the same pairs of positions.
83. The mixture of claim 82, wherein the first antibody is an IgG1,
IgG2, or IgG4 antibody, and the second antibody is an IgG1, IgG2,
or IgG4 antibody.
84. The mixture of claim 83, wherein the mixture comprises a third
major antibody species comprising one chain of the HC1, one chain
of the LC1, one chain of the HC2, and one chain of the LC2.
85. The mixture of claim 83, wherein the first antibody and/or the
second antibody comprise(s) (a) charge pair(s), wherein one amino
acid of the charge pair(s) is in the CH1 and the other amino acid
of the charge pair(s) is in the CL of the antibody or antibodies
comprising the charge pair, and wherein: (1) if the antibody or
antibodies comprising the charge pair(s) is (are) (an) IgG1
antibody or antibodies, then the charge pair(s) is (are) selected
from the group consisting of the following pairs of residues, which
are specified by their the heavy and light chain positions,
respectively, and the amino acids occupying those positions: 147D/E
and 124K/R; 147K/R and 124D/E; 147D/E and 129K/R; 147K/R and
129D/E; 147D/E and 131K/R; 147K/R and 131D/E; 147D/E and 180K/R;
147K/R and 180D/E; 168D/E and 164K/R; 168K/R and 164D/E; 168D/E and
167K/R; 168K/R and 167D/E; 168D/E and 174K/R; and 168K/R and
174D/E; (2) if the antibody or antibodies comprising the charge
pair(s) is (are) (an) IgG2 antibody or antibodies, then the charge
pair(s) is (are) selected from the group consisting of the
following pairs of residues, which are specified by their the heavy
and light chain positions, respectively, and the amino acids
occupying those positions: 147D/E and 131K/R; and 147K/R and
131D/E; and (3) if the antibody or antibodies comprising the charge
pair(s) is (are) (an) IgG4 antibody or antibodies, then the charge
pair(s) is (are) selected from the group consisting of the
following pairs of residues, which are specified by their the heavy
and light chain positions, respectively, and the amino acids
occupying those positions: 133D/E and 117K/R; 133K/R and 117D/E;
137K/R and 114D/E; 137D/E and 114K/R; 137K/R and 116D/E; 137D/E and
116K/R; 147D/E and 124K/R; 147K/R and 124D/E; 147D/E and 129K/R;
147K/R and 129D/E; 147D/E and 131K/R; 147K/R and 131D/E; 147D/E and
178K/R; 147K/R and 178D/E; 147D/E and 180K/R; 147K/R and 180D/E;
168D/E and 164K/R; 168K/R and 164D/E; 168D/E and 167K/R; 168K/R and
167D/E; 168D/E and 173K/R; 168K/R and 173D/E; 168D/E and 174K/R;
and 168K/R and 174D/E.
86. The mixture of claim 85, wherein the pair of contacting
cysteine residues in the first or second antibody is at positions
170 of HC1 and 162 of LC1 or positions 170 of HC2 and 162 of LC2,
and wherein the amino acids comprising the charge pair in the first
or second antibody are 147D/E in the heavy chain of the first or
second antibody and 131R/K in the cognate light chain.
87. The mixture of claim 83, wherein the first antibody comprises
one or more partner-directing alteration(s) and the second does
not; and wherein (1) if the first antibody is an IgG1 antibody,
then the cysteine at position 220 in the HC1 is substituted with
another amino acid, and the cysteine at position 214 in the LC1 is
substituted with another amino acid, and (2) if the first antibody
is an IgG2 or IgG4 antibody, then the cysteine at position 131 in
the HC1 is substituted with another amino acid, and the cysteine at
position 214 in the LC1 is substituted with another amino acid.
88. The mixture of claim 87, wherein (a) the first antibody is an
IgG1 antibody, and it comprises the alterations C220G/A/S in the
HC1 and C214S/A/G in the LC1; or (b) the first antibody is an IgG2
or IgG4 antibody, and it comprises the alterations C131SING in the
HC1 and C214S/A/G in the LC1.
89. The mixture of claim 88, wherein the first antibody is an IgG1
antibody and comprises 147D, 170C, 173C, and 220G in the HC1 and
131K, 160C, 162C, and 214S in the LC1; or wherein the first
antibody is an IgG2 antibody and comprises 147D, 170C, 173C, and
131S/A/G in the HC1 and 131K, 160C, 162C, and 214S in the LC1.
90. The mixture of claim 88, wherein the first antibody comprises
399R and 409E and the second antibody comprises 409R.
91. The mixture of claim 82, wherein both the first and second
antibodies comprise one or more partner-directing alteration(s),
the HC1 and the HC2 comprise a charged amino acid at an HC residue
at the same position in both the HC1 and the HC2, the charged amino
acid at the HC residue in the HC1 is opposite in charge to the
charged amino acid at the HC residue at the same position in the
HC2, the LC1 and the LC2 comprise a charged amino acid at an LC
residue at the same position in both the LC1 and the LC2, the LC
residue contacts the HC residue, the charged amino acid at the LC
residue in the LC1 is opposite in charge to the charged amino acid
at the LC residue in the LC2, and the charged amino acid at the LC
residue in the LC1 is opposite in charge to the amino acid at the
HC residue in the HC1.
92. The mixture of claim 91, wherein: both the HC1 and the HC2
comprise charged amino acids at positions 44, 105, 147, and 168;
and both the LC1 and the LC2 comprise charged amino acids at
positions 43, 100, 131, and 174.
93. The mixture of claim 91, wherein one of the first and second
antibodies comprises 399R and 409E and the other comprises
409R.
94. The mixture of claim 83, wherein if the HC1 and/or the HC2 is
an IgG4 HC, then the IgG4 HC(s) comprise(s) 228P.
95. The mixture of claim 83, wherein the in vivo half lives of the
first and second antibodies differ by at least one week, and
wherein the antibody with the shorter in vivo half life comprises
at least one of the following alterations: M252A, M252L, M252S,
M252R, R255K or H435R.
96. The mixture of claim 82, wherein (a) one of the first and
second antibodies binds to human CTLA4 and the other binds to human
PD1, (b) both the first and second antibodies bind to human HER2,
but they do not compete for binding to HER2, (c) one of the first
and second antibodies binds to human LAG3 and the other binds to
human PD1, (d) one of the first and second antibodies binds to
human GITR and the other binds to human PD1, (d) one of the first
and second antibodies binds to human VEGF and the other binds to
human PD1, (f) one of the first and second antibodies binds to
human CSFR1a and the other binds to human PD1, (g) one of the first
and second antibodies binds to human OX40 and the other binds to
human PD1, (h) one of the first and second antibodies binds to
human TIGIT and the other binds to human PD1, (i) one of the first
and second antibodies binds to human CTLA4 and the other binds to
human PDL1, (j) one of the first and second antibodies binds to
human VEGF and the other binds to human PDL1, (k) one of the first
and second antibodies binds to human OX40 and the other binds to
human PDL1, (l) one of the first and second antibodies binds to
human CSFR1a and the other binds to human PDL1, (m) one of the
first and second antibodies binds to human TIGIT and the other
binds to human PDL1, (n) one of the first and second antibodies
binds to human Tim3 and the other binds to human PDL1, (o) one of
the first and second antibodies binds to human CTLA4 and the other
binds to human VEGF, (p) one of the first and second antibodies
binds to human CTLA4 and the other binds to human 41BB, (q) one of
the first and second antibodies binds to human CD20 and the other
binds to human CD37, (r) one of the first and second antibodies
binds to human ANG2 and the other binds to human VEGF, (s) one of
the first and second antibodies binds to human TNF and the other
binds to human IL17a, (t) one of the first and second antibodies
binds to human CD38 and the other binds to human CD138, (u) one of
the first and second antibodies binds to human EGFR and the other
binds to human HER2, (v) one of the first and second antibodies
binds to human EGFR and the other binds to human HER3, (w) one of
the first and second antibodies binds to human MET and the other
binds to human VEGF (x) one of the first and second antibodies
binds to human MET and the other binds to human EGFR, (y) one of
the first and second antibodies binds to human TSLP and the other
binds to human IL33, (z) one of the first and second antibodies
binds to human IL4 and the other binds to human IL13, (aa) one of
the first and second antibodies binds to human CSF1R protein and
the other binds to a human SIRP-alpha protein, (bb) one of the
first and second antibodies binds to human PD1 protein and the
other binds to a human SIRP-alpha protein, or (cc) one of the first
and second antibodies binds to human PD1 and the other binds to
human CCR8.
97. One or more nucleic acid(s) encoding a mixture of antibodies
comprising two and not more than three different major antibody
species, which include (a) a first antibody comprising two chains
of a first heavy chain (HC1), each having the same first amino acid
sequence, and two chains of a first light chain (LC1), each having
the same second amino acid sequence, and (b) a second antibody
comprising two chains of a second heavy chain (HC2), each having
the same third amino acid sequence, and two chains of a second
light chain (LC2), each having the same fourth amino acid sequence,
wherein: (1) the first and second antibodies are full-length human
and/or humanized IgG antibodies; (2) the HC1 and the HC2 have
different amino acid sequences, and the LC1 and the LC2 have
different amino acid sequences; (3) the first heavy chain constant
(CH1) and the light chain constant (CL) domains of the first
antibody and/or the second antibody comprise one or more
partner-directing alterations that create one or more pairs of
contacting cysteine residues, wherein one cysteine residue of the
pair(s) is in the CH1 domain and the other is in the CL domain of
the antibody or antibodies comprising the pair(s) of contacting
cysteine residues, wherein (A) if the first and/or second antibody
comprising the contacting cysteine residues is (are) (an) IgG1
antibody or antibodies, then the pair(s) of heavy chain and light
chain positions, respectively, of the pair(s) of contacting
cysteine residues in the IgG1 antibody or antibodies is (are)
selected from the group consisting of: positions 126 and 124;
positions 128 and 118; positions 133 and 117; positions 133 and
209; positions 134 and 116; positions 168 and 174; positions 170
and 162; positions 170 and 176; positions 173 and 160; and
positions 183 and 176, (B) if the first and/or second antibody
comprising the contacting cysteine residues is (are) (an) IgG2
antibody or antibodies, then the pair(s) of heavy chain and light
chain positions, respectively, of the pair(s) of contacting
cysteine residues in the IgG2 antibody or antibodies is (are)
selected from the group consisting of: positions 170 and 162; and
positions 173 and 160, and (C) if the first and/or second antibody
comprising the contacting cysteine residues is (are) (an) IgG4
antibody or antibodies, then the pair(s) of heavy chain and light
chain positions, respectively, of the pair(s) of contacting
cysteine residues in the IgG4 antibody or antibodies is (are)
selected from the group consisting of: positions 126 and 121;
positions 126 and 124; positions 127 and 121; positions 128 and
118; positions 168 and 174; positions 170 and 162; and positions
173 and 162; and (4) if both the first and second antibodies
comprise a pair of contacting cysteine residues as recited in (3)
above, then the first and second antibodies do not comprise
contacting cysteine residues at the same pairs of positions.
98. The nucleic acid(s) of claim 97, wherein the first antibody is
an IgG1, IgG2, or IgG4 antibody and the second antibody is an IgG1,
IgG2, or IgG4 antibody.
99. The nucleic acid(s) of claim 97, which include a mammalian
expression vector or a viral vector, and wherein the viral vector
is selected from the group consisting of: an adenovirus vector, an
adeno-associated virus (AAV) vector, a retrovirus vector, a
vaccinia virus vector, a modified vaccinia virus Ankara (MVA)
vector, a herpes virus vector, a lentivirus vector, and a poxvirus
vector.
100. A method of making a mixture of antibodies comprising the
steps of: (a) introducing one or more nucleic acid(s) encoding the
mixture of antibodies into a host cell line, (b) culturing the host
cell line expressing the mixture of antibodies in a culture medium,
and (c) recovering the mixture of antibodies from the cell mass or
the culture medium, wherein (1) the mixture comprises (A) a first
antibody comprising two chains of a first heavy chain (HC1) each
having the same first amino acid sequence and two chains of a first
light chain (LC1) each having the same second amino acid sequence
and (B) a second antibody comprising two chains of a second heavy
chain (HC2) each having the same third amino acid sequence and two
chains of a second light chain (LC2) each having the same fourth
amino acid sequence, (2) the first and second antibodies are
full-length human and/or humanized IgG antibodies; (3) the HC1 and
the HC2 have different amino acid sequences, and the LC1 and the
LC2 have different amino acid sequences; and (4) the mixture
comprises not more than three different major species of
full-length IgG antibodies.
101. The method of claim 100, wherein: (a) the nucleic acid(s)
encoding the HC1, HC2, LC1, and LC2 was (were) introduced at the
same time; (b) the nucleic acid(s) encoding the HC1 and LC1 was
(were) introduced before the nucleic acid(s) encoding the HC2 and
LC2; or (c) the nucleic acid(s) encoding the HC2 and LC2 was (were)
introduced before the nucleic acid(s) encoding the HC1 and LC1.
102. The method of a claim 101, wherein the host cell line is a
mammalian cell line.
103. A host cell line that contains nucleic acid(s) encoding a
mixture of antibodies comprising two and not more than three
different major antibody species, which includes (a) a first
antibody comprising two chains of a first heavy chain (HC1), each
having the same first amino acid sequence, and two chains of a
first light chain (LC1), each having the same second amino acid
sequence, and (b) a second antibody comprising two chains of a
second heavy chain (HC2), each having the same third amino acid
sequence, and two chains of a second light chain (LC2), each having
the same fourth amino acid sequence, wherein: (1) the first and
second antibodies are full-length human and/or humanized IgG
antibodies; (2) the HC1 and the HC2 have different amino acid
sequences, and the LC1 and the LC2 have different amino acid
sequences; (3) the first heavy chain constant (CH1) and the light
chain constant (CL) domains of the first antibody and/or the second
antibody comprise one or more partner-directing alterations that
create one or more pairs of contacting cysteine residues, wherein
one cysteine residue of the pair(s) is in the CH1 domain and the
other is in the CL domain of the antibody or antibodies comprising
the pair(s) of contacting cysteine residues, wherein (A) if the
first and/or second antibody comprising the contacting cysteine
residues is (are) (an) IgG1 antibody or antibodies, then the
pair(s) of heavy chain and light chain positions, respectively, of
the pair(s) of contacting cysteine residues in the IgG1 antibody or
antibodies is (are) selected from the group consisting of:
positions 126 and 124; positions 128 and 118; positions 133 and
117; positions 133 and 209; positions 134 and 116; positions 168
and 174; positions 170 and 162; positions 170 and 176; positions
173 and 160; and positions 183 and 176, (B) if the first and/or
second antibody comprising the contacting cysteine residues is
(are) (an) IgG2 antibody or antibodies, then the pair(s) of heavy
chain and light chain positions, respectively, of the pair(s) of
contacting cysteine residues in the IgG2 antibody or antibodies is
(are) selected from the group consisting of: positions 170 and 162;
and positions 173 and 160, and (C) if the first and/or second
antibody comprising the contacting cysteine residues is (are) (an)
IgG4 antibody or antibodies, then the pair(s) of heavy chain and
light chain positions, respectively, of the pair(s) of contacting
cysteine residues in the IgG4 antibody or antibodies is (are)
selected from the group consisting of: positions 126 and 121;
positions 126 and 124; positions 127 and 121; positions 128 and
118; positions 168 and 174; positions 170 and 162; and positions
173 and 162; and (4) if both the first and second antibodies
comprise a pair of contacting cysteine residues as recited in (3)
above, then the first and second antibodies do not comprise
contacting cysteine residues at the same pairs of positions.
104. The host cell line of claim 103, which is a CHO cell line.
105. A method of treating a disease comprising administering to a
patient having the disease a mixture of antibodies or (a) nucleic
acid(s) encoding the mixture of antibodies, wherein: (a) the
disease is a cancer, a metabolic disease, an infectious disease, or
an autoimmune or inflammatory disease; (b) the mixture of
antibodies comprises not more than three major species of
antibodies including (1) a first antibody comprising two chains of
a first heavy chain (HC1) each having the same first amino acid
sequence and two chains of a first light chain (LC1) each having
the same second amino acid sequence and (2) a second antibody
comprising two chains of a second heavy chain (HC2) each having the
same third amino acid sequence and two chains of a second light
chain (LC2) each having the same fourth amino acid sequence; (c)
the first and second antibodies are full-length human and/or
humanized IgG antibodies, the HC1 and the HC2 have different amino
acid sequences, the LC1 and the LC2 have different amino acid
sequences, and the mixture comprises not more than three different
major species of full-length IgG antibodies; (d) the CH1 and CL
domains of the first antibody and/or the second antibody comprise
one or more partner-directing alters creating a pair of contacting
cysteine residues, wherein one cysteine residue of the pair is in
the CH1 domain and the other is in the CL domain of the antibody or
antibodies comprising the pair(s) of contacting cysteine residues;
(e) if the first and/or second antibody comprising the contacting
cysteine residues is (are) (an) IgG1 antibody or antibodies, then
the pair(s) of heavy chain and light chain positions, respectively,
of the pair(s) of contacting cysteine residues in the IgG1 antibody
or antibodies is (are) selected from the group consisting of:
positions 126 and 124; positions 128 and 118; positions 133 and
117; positions 133 and 209; positions 134 and 116; positions 168
and 174; positions 170 and 162; positions 170 and 176; positions
173 and 160; and positions 183 and 176; (f) if the first and/or
second antibody comprising the contacting cysteine residues is
(are) (an) IgG2 antibody or antibodies, then the pair(s) of heavy
chain and light chain positions, respectively, of the pair(s) of
contacting cysteine residues in the IgG2 antibody or antibodies is
(are) selected from the group consisting of: positions 170 and 162;
and positions 173 and 160, (g) if the first and/or second antibody
comprising the contacting cysteine residues is (are) (an) IgG4
antibody or antibodies, then the pair(s) of heavy chain and light
chain positions, respectively, of the pair(s) of contacting
cysteine residues in the IgG4 antibody or antibodies is (are)
selected from the group consisting of: positions 126 and 121;
positions 126 and 124; positions 127 and 121; positions 128 and
118; positions 168 and 174; positions 170 and 162; and positions
173 and 162; and (h) if both the first and second antibodies
comprise a pair of contacting cysteine residues as recited in
(d)-(g), then the first and second antibodies do not comprise
contacting cysteine residues at the same pairs of positions.
106. The method of claim 105, wherein the disease is a cancer and
wherein (1) the first antibody in the mixture of antibodies binds
to human CTLA4 and the second antibody in the mixture of antibodies
binds to human PD1, or vice versa, (2) both the first and second
antibodies in the mixture of antibodies bind to human HER2, but
they do not compete for binding to HER2, or (3) the first antibody
in the mixture of antibodies binds to human CD20 and the second
antibody in the mixture of antibodies binds to human CD37, or vice
versa.
107. The method of claim 106, wherein the disease is a cancer and
the patient has a tumor, and wherein the method comprises injecting
the mixture of antibodies or the nucleic acid(s) encoding the
mixture of antibodies into the tumor.
Description
FIELD
[0001] The compositions and methods described herein are in the
field of recombinant antibodies and methods for their
production.
BACKGROUND
[0002] Recombinant monoclonal antibodies have emerged as a very
successful class of biological drugs for the treatment of a variety
of different diseases during the past two decades. They have been
used both with and without the co-administration of small
molecule-based drugs. Due to the biological complexity of some
diseases, antibody mixtures or bispecific antibodies that target
more than one antigen or epitope can be more effective than single
antibodies in treating certain conditions. See, e.g., Lindzen et
al., (2010), Proc. Natl. Acad. Sci. 107(28): 12550-12563; Nagorsen
and Baeuerle (2011), Exp. Cell Res. 317(9): 1255-60.
[0003] Antibody mixtures allow flexibility in dosing of two
different binding entities. This is not true for an IgG bispecific
antibody because each of the two binding entities is present in the
same amount since the binding entities are part of the same
molecule. This may not be optimal in cases where different doses of
each binding entity are desirable in a treatment regimen. See,
e.g., Wolchok et al. (2013), New Engl. J. Med. 369(2): 122-133.
Similarly, both binding entities will have the same in vivo half
life in a bispecific antibody, which may not be optimal in some
situations. Therapeutics including mixtures of two or more
individual antibodies could allow different doses of antibodies
comprising two different binding entities to be administered, and
the antibodies could have different pharmacokinetic properties.
[0004] The manufacture of recombinant antibody mixtures can be
accomplished through the separate production of each antibody from
individual host cell lines and the mixture of multiple antibodies
post production. Single batch production of multiple antibodies by
mixing and co-culturing host cells expressing different antibodies
has provided a less costly alternative to this approach. Rasmussen
et al. (2012), Arch Biochem Biophys. 526: 139-45. Other simple and
relatively inexpensive production processes for producing two or
more antibodies using a single process are needed.
SUMMARY
[0005] The compositions and methods set forth herein are described
below in a numbered series of items.
[0006] 1. A mixture of antibodies comprising two major antibody
species including
[0007] (a) a first antibody comprising two copies of a first heavy
chain (HC1) having the same amino acid sequence and two copies of a
first light chain (LC1) having the same amino acid sequence and
[0008] (b) a second antibody comprising two copies of a second
heavy chain (HC2) having the same amino acid sequence and two
copies of a second light chain (LC2) having the same amino acid
sequence,
[0009] wherein the first and second antibodies are full-length
primate IgG antibodies,
[0010] wherein the HC1 and the HC2 have different amino acid
sequences,
[0011] wherein the LC1 and the LC2 have different amino acid
sequences,
[0012] wherein the mixture comprises not more than three different
major species of full-length antibodies, and
[0013] wherein the mixture has been produced by a single host cell
line into which DNA encoding the HC1, HC2, LC1, and LC2 has been
introduced.
[0014] 2. The mixture of item 1, wherein the HC1 and HC2 are human
IgG HCs, wherein each is of any one of the following isotypes:
IgG1, IgG2, IgG3, or IgG4.
[0015] 3. The mixture of item 1 or 2, wherein
[0016] the HC1 and/or HC2 contain(s) at least one
LC-partner-directing alteration, and
[0017] the LC1 and/or LC2 contain(s) at least one
HC-partner-directing alteration at an amino acid contacting the
amino acid at which the LC-partner-directing alteration(s) occur(s)
in the HC1 and/or HC2, respectively, or contacting a charged amino
acid or cysteine present in the HC1 and/or HC2, respectively.
[0018] 4. The mixture of item 3,
[0019] wherein the HC1 and/or HC2 comprise at least one
LC-partner-directing alteration at a heavy chain (HC) residue,
wherein the LC partner-directing alteration(s) cause(s) the HC1 and
HC2 to comprise a charged amino acid at the same HC residue in both
the HC1 and HC2 amino acid sequences, wherein the charged amino
acid at the HC residue in the HC1 is opposite in charge to the
charged amino acid at the HC residue in the HC2, and
[0020] wherein the LC1 and/or LC2 comprise at least one
HC-partner-directing alteration at a light chain (LC) residue
contacting the HC residue, wherein the HC-partner-directing
alteration(s) causes the LC1 and LC2 to comprise a charged amino
acid at the same LC residue in the both the LC1 and LC2 amino acid
sequences, wherein the charged amino acid at the LC residue in the
LC1 is opposite in charge to the charged amino acid at the LC
residue in the LC2, and the charged amino acid at the LC residue in
the LC1 is opposite in charge to the amino at the HC residue in the
HC1.
[0021] 5. The mixture of item 3 or 4, wherein the first and second
antibodies comprise one or more of the sets of alterations selected
from the group consisting of:
[0022] (a) the set of alterations wherein positions 44 and 105 in
both the HC1 and HC2 and 43 and 100 in both the LC1 and LC2 are
each occupied by a charged amino acid, the amino acids at positions
44 and 105 in the HC1 are each opposite in charge from those at the
same sites in the HC2, the amino acids at positions 43 and 100 in
the LC1 are each opposite in charge from those at the same sites in
LC2, the amino acids at position 44 in the HC1 and HC2 are opposite
in charge to the amino acids at position 100 in the LC1 and LC2,
respectively, and the amino acids at position 105 in the HC1 and
HC2 are opposite in charge to the amino acids at position 43 in the
LC1 and LC2, respectively;
[0023] (b) the set of alterations wherein positions 147 in the HC1
and HC2 and 131 in the LC1 and LC2 are each occupied by a charged
amino acid, the amino acid at position 147 in the HC1 is opposite
in charge from that at position 147 in the HC2, the amino acid at
position 131 in the LC1 is opposite in charge from that at position
131 in the LC2, and the amino acids at position 147 in the HC1 and
HC2 are opposite in charge to the amino acids at position 131 in
the LC1 and LC2, respectively;
[0024] (c) the set of alterations wherein positions 168 in the HC1
and HC2 and 174 in the LC1 and LC2 are each occupied by a charged
amino acid, the amino acid at position 168 the HC1 is opposite in
charge from that at position 168 in the HC2, the amino acid at
position 174 in the LC1 is opposite in charge from that at position
174 in the LC2, and the amino acids at position 168 in the HC1 and
HC2 are opposite in charge to the amino acids at position 174 in
the LC1 and LC2, respectively; and
[0025] (d) the set of alterations wherein positions 181 in the HC1
and HC2 and 178 in the LC1 and LC2 are each occupied by a charged
amino acid, the amino acid at position 181 in the HC1 is opposite
in charge from that at position 181 in the HC2, the amino acid at
position 178 in the LC1 is opposite in charge from that at position
178 in the LC2, and the amino acids at position 181 in the HC1 and
HC2 are opposite in charge to the amino acids at position 178 in
the LC1 and LC2, respectively.
[0026] 6. The mixture of any one of items 1 to 5, wherein the first
and second antibodies comprise one or more of the sets of
alterations selected from the group consisting of:
[0027] (a) the set of alterations wherein the HC1 comprises 44E/D,
the LC1 comprises 100R/K, the HC2 comprises 44R/K, and the LC2
comprises 100D/E; or the HC2 comprises 44E/D, the LC2 comprises
100R/K, the HC1 comprises 44R/K, and the LC1 comprises 100D/E;
[0028] (b) the set of alterations wherein the HC1 comprises 105E/D,
the LC1 comprises 43R/K, the HC2 comprises 105R/K, and the LC2
comprises 43D/E; or the HC2 comprises 105E/D, the LC2 comprises
43R/K, the HC1 comprises 105R/K, and the LC1 comprises 43D/E;
[0029] (c) the set of alterations wherein the HC1 comprises 147R/K,
the LC1 comprises 131E/D, the HC2 comprises 147E/D, and the LC2
comprises 131R/K; or the HC2 comprises 147R/K, the LC2 comprises
131E/D, the HC1 comprises 147E/D, and the LC1 comprises 131R/K;
[0030] (d) the set of alterations wherein the HC1 comprises 168E/D,
the LC1 comprises 174R/K, the HC2 comprises 168R/K, and the LC2
comprises 174D/E; or the HC2 comprises 168E/D, the LC2 comprises
174R/K, the HC1 comprises 168R/K, and the LC1 comprises 174D/E;
and
[0031] (e) the set of alterations wherein the HC1 comprises 181E/D,
the LC1 comprises 178R/K, the HC2 comprises 181R/K, and the LC2
comprises 178D/E; or the HC2 comprises 181E/D, the LC2 comprises
178R/K, the HC1 comprises 181R/K, and the LC1 comprises 178D/E.
[0032] 7. The mixture of item 6, wherein the first and second
antibodies comprise one or more of the sets of alterations selected
from the group consisting of:
[0033] (a) the set of alterations wherein the HC1 comprises 44D and
105R, the LC1 comprises 43D and 100R, the HC2 comprises 44R and
105D, and the LC2 comprises 43R and 100D; or the HC2 comprises 44D
and 105R, the LC2 comprises 43D and 100R, the HC1 comprises 44R and
105D, and the LC1 comprises 43R and 100D;
[0034] (b) the set of alterations wherein the HC1 comprises 147R,
the LC1 comprises 131D, the HC2 comprises 147D, and the LC2
comprises 131R; or the HC2 comprises 147R, the LC2 comprises 131D,
the HC1 comprises 147D, and the LC1 comprises 131R;
[0035] (c) the set of alterations wherein the HC1 comprises 168D,
the LC1 comprises 174R, the HC2 comprises 168R, and the LC2
comprises 174D; or the HC2 comprises 168D, the LC2 comprises 174R,
the HC1 comprises 168R, and the LC1 comprises 174D; and
[0036] (d) the set of alterations wherein the HC1 comprises 181D,
the LC1 comprises 178R, the HC2 comprises 181R, and the LC2
comprises 178D; or the HC2 comprises 181D, the LC2 comprises 178R,
the HC1 comprises 181R, and the LC1 comprises 178D.
[0037] 8. The mixture of item 7, wherein the first and second
antibodies comprise one or more of the sets of alterations selected
from the group consisting of:
[0038] (a) the set of alterations wherein the HC1 comprises G44D
and Q105R, the LC1 comprises A/V43D and Q/G100R, the HC2 comprises
G44R and Q105D, and the LC2 comprises A/V43R and Q/G100D; or the
HC2 comprises G44D and Q105R, the LC2 comprises AA/43D and Q/G100R,
the HC1 comprises G44R and Q105D, and the LC1 comprises A/V43R and
Q/G100D;
[0039] (b) the set of alterations wherein the HC1 comprises K147R,
the LC1 comprises S131D, the HC2 comprises K147D, and the LC2
comprises S131R; or the HC2 comprises K147R, the LC2 comprises
S131D, the HC1 comprises K147D, and the LC1 comprises S131R;
[0040] (c) the set of alterations wherein the HC1 comprises H168D,
the LC1 comprises S174R, the HC2 comprises H168R, and the LC2
comprises S174D; or the HC2 comprises H168D, the LC2 comprises
S174R, the HC1 comprises H168R, and the LC1 comprises S174D;
and
[0041] (d) the set of alterations wherein the HC1 comprises S181D,
the LC1 comprises T178R, the HC2 comprises S181R, and the LC2
comprises T178D; or the HC2 comprises S181D, the LC2 comprises
T178R, the HC1 comprises S181R, and the LC1 comprises T178D.
[0042] 9. The mixture of any one of items 1 to 8, wherein the first
and/or the second antibody comprise one or more of the pairs of
alterations selected from the group consisting of:
[0043] 126C in the HC1 and 121C in the LC1, or 126C in the HC2 and
121C in the LC2;
[0044] 126C in the HC1 and 124C in the LC1, or 126C in the HC2 and
124C in the LC2;
[0045] 127C in the HC1 and 121C in the LC1, or 127C in the HC2 and
121C in the LC2;
[0046] 128C in the HC1 and 118C in the LC1, or 128C in the HC2 and
118C in the LC2;
[0047] 133C in the HC1 and 117C in the LC1, or 133C in the HC2 and
117C in the LC2;
[0048] 133C in the HC1 and 209C in the LC1, or 133C in the HC2 and
209C in the LC2;
[0049] 134C in the HC1 and 116C in the LC1, or 134C in the HC2 and
116C in the LC2;
[0050] 141C in the HC1 and 116C in the LC1, or 141C in the HC2 and
116C in the LC2;
[0051] 168C in the HC1 and 174C in the LC1, or 168C in the HC2 and
174C in the LC2;
[0052] 170C in the HC1 and 162C in the LC1, or 170C in the HC2 and
162C in the LC2;
[0053] 170C in the HC1 and 176C in the LC1, or 170C in the HC2 and
176C in the LC2;
[0054] 173C in the HC1 and 160C in the LC1, or 173C in the HC2 and
160C in the LC2;
[0055] 173C in the HC1 and 162C in the LC1, or 173C in the HC2 and
162C in the LC2; and
[0056] 183C in the HC1 and 176C in the LC1, or 183C in the HC2 and
176C in the LC2.
[0057] 10. The mixture of any one of items 1 to 9, wherein the
first and second antibodies comprise one or more of the sets of
alterations selected from the group consisting of:
[0058] (1) positions 170 and 183 in the HC1 and HC2, respectively,
and 162 and 176 in the LC1 and LC2, respectively, are each
substituted with cysteine; or positions 170 and 183 in the HC2 and
HC1, respectively, and 162 and 176 in the LC2 and LC1,
respectively, are each substituted with cysteine;
[0059] (2) positions 173 and 170 in the HC1 and HC2, respectively,
and 160 and 162 in the LC1 and LC2, respectively, are each
substituted with cysteine; or positions 173 and 170 in the HC2 and
HC1, respectively, and 160 and 162 in the LC2 and LC1,
respectively, are each substituted with cysteine;
[0060] (3) positions 173 and 183 in the HC1 and HC2, respectively,
and 160 and 176 in the LC1 and LC2, respectively, are each
substituted with cysteine; or positions 173 and 183 in the HC2 and
HC1, respectively, and 160 and 176 in the LC2 and LC1,
respectively, are each substituted with cysteine;
[0061] (4) positions 170 in HC1 and the HC2 and 162 and 176 in the
LC1 and LC2, respectively, are each substituted with cysteine; or
positions 170 in the HC2 and HC1 and 162 and 176 in the LC2 and
LC1, respectively, are each substituted with cysteine;
[0062] (5) positions 170 and 173 in the HC1 and HC2, respectively,
and the 162 and 160 in LC1 and LC2, respectively, are each
substituted with cysteine; or positions 170 and 173 in the HC2 and
HC1, respectively, and 162 and 160 in the LC2 and LC1,
respectively, are each substituted with cysteine;
[0063] (6) positions 173 and 170 in the HC1 and HC2, respectively,
and 162 and 176 in the LC1 and LC2, respectively, are each
substituted with cysteine; or positions 173 and 170 in the HC2 and
HC1, respectively, and 162 and 176 in the LC2 and LC1,
respectively, are each substituted with cysteine;
[0064] (7) positions 173 in the HC1 and HC2 and 162 and 160 in the
LC1 and LC2, respectively, are each substituted with cysteine; or
positions 173 in the HC2 and HC1 and 162 and 160 in the LC2 and
LC1, respectively, are each substituted with cysteine;
[0065] (8) positions 183 and 173 in the HC1 and HC2, respectively,
and 176 and 160 in the LC1 and LC2, respectively, are each
substituted with cysteine; or positions 183 and 173 in the HC2 and
HC1, respectively, and 176 and 160 in the LC2 and LC1,
respectively, are each substituted with cysteine;
[0066] (9) positions 170 and 173 in the HC1 and HC2, respectively,
and 176 and 160 in the LC1 and LC2, respectively, are each
substituted with cysteine; or positions 170 and 173 in the HC2 and
HC1, respectively, and 176 and 160 in the LC2 and LC1,
respectively, are each substituted with cysteine; and
[0067] (10) positions 170 and 183 in the HC1 and HC2, respectively
and 176 in the LC1 and LC2 are each substituted with cysteine; or
positions the 170 and 183 in HC2 and HC1, respectively and 176 in
the LC2 and LC1 are each substituted with cysteine.
[0068] 11. The mixture of any one of items 1 to 10, wherein the
first and second antibodies comprise one or more of the sets of
alterations selected from the group consisting of:
[0069] (1) the HC1 comprises F170C, the LC1 comprises S162C, the
HC2 comprises S183C, and the LC2 comprises S176C; or the HC2
comprises F170C, the LC2 comprises S162C, the HC1 comprises S183C,
and the LC1 comprises S176C;
[0070] (2) the HC1 comprises V173C, the LC1 comprises 0160C, the
HC2 comprises F170C, and the LC2 comprises S162C; or the HC2
comprises V173C, the LC2 comprises 0160C, the HC1 comprises F170C,
and the LC1 comprises S162C;
[0071] (3) the HC1 comprises V173C, the LC1 comprises 0160C, the
HC2 comprises S183C, and the LC2 comprises S176C; or the HC2
comprises V173C, the LC2 comprises 0160C, the HC1 comprises S183C,
and the LC1 comprises S176C;
[0072] (4) the HC1 comprises F170C, the LC1 comprises S162C, the
HC2 comprises F170C, and the LC2 comprises S176C; or the HC2
comprises F170C, the LC2 comprises S162C, the HC1 comprises F170C,
and the LC1 comprises S176C;
[0073] (5) the HC1 comprises F170C, the LC1 comprises S162C, the
HC2 comprises V173C, and the LC2 comprises 0160C; or the HC2
comprises F170C, the LC2 comprises S162C, the HC1 comprises V173C,
and the LC1 comprises 0160C;
[0074] (6) the HC1 comprises V173C, the LC1 comprises S162C, the
HC2 comprises F170C, and the LC2 comprises S176C; or the HC2
comprises V173C, the LC2 comprises S162C, the HC1 comprises F170C,
and the LC1 comprises S176C;
[0075] (7) the HC1 comprises V173C, the LC1 comprises S162C, the
HC2 comprises V173C, and the LC2 comprises 0160C; or the HC2
comprises V173C, the LC2 comprises S162C, the HC1 comprises V173C,
and the LC1 comprises 0160C;
[0076] (8) the HC1 comprises S183C, the LC1 comprises S176C, the
HC2 comprises V173C, and the LC2 comprises 0160C; or the HC2
comprises S183C, the LC2 comprises S176C, the HC1 comprises V173C,
and the LC1 comprises 0160C;
[0077] (9) the HC1 comprises F170C, the LC1 comprises S176C, the
HC2 comprises V173C, and the LC2 comprises 0160C; or the HC2
comprises F170C, the LC2 comprises S176C, the HC1 comprises V173C,
and the LC1 comprises 0160C; and
[0078] (10) the HC1 comprises F170C, the LC1 comprises S176C, the
HC2 comprises S183C, and the LC2 comprises S176C; or the HC2
comprises F170C, the LC2 comprises S176C, the HC1 comprises S183C,
and the LC1 comprises S176C.
[0079] 12. The mixture of any one of items 1 to 11, which comprises
three major antibody species.
[0080] 13. The mixture of any one of items 1 to 11, which comprises
not more than two major antibody species.
[0081] 14. The mixture of item 13,
[0082] wherein the HC1 and/or HC2 contain(s) one or more
alteration(s) that disfavor(s) heterodimers.
[0083] 15. The mixture of item 14, wherein
[0084] (a) (1) the HC1 is an IgG1 HC, the HC2 is an IgG4 HC, the
HC1 comprises alterations that disfavor heterodimers at positions
399 and 409, and the HC2 does not contain an alteration that
disfavors heterodimers; or (2) the HC2 is an IgG1 HC, the HC1 is an
IgG4 HC, the HC2 comprises alterations that disfavor heterodimers
at positions 399 and 409, and the HC1 does not contain an
alteration that disfavors heterodimers;
[0085] (b) (1) the HC1 and HC2 are IgG1 HCs, the HC1 comprises
alterations that disfavor heterodimers at positions 399 and 409,
and the HC2 comprises the alteration K409R; or (2) the HC1 and HC2
are IgG1 HCs, the HC2 comprises alterations that disfavor
heterodimers at positions 399 and 409, and the HC1 comprises the
alteration K409R;
[0086] (c) (1) the HC1 is an IgG1 HC, the HC2 is an IgG4 HC, and
the HC1 comprises alterations that disfavor heterodimers at
positions 390 and 400; or (2) the HC1 is an IgG1 HC, the HC2 is an
IgG4 HC, and the HC2 comprises alterations that disfavor
heterodimers at positions 390 and 400;
[0087] (d) (1) the HC1 and HC2 are IgG1 HCs, and the HC1 comprises
alterations that disfavor heterodimers at positions 390 and 400; or
(2) the HC1 and HC2 are IgG1 HCs, and the HC2 comprises alterations
that disfavor heterodimers at positions 390 and 400;
[0088] (e) (1) the HC1 is an IgG1 HC, the HC2 is an IgG4 HC, the
HC1 comprises alterations that disfavor heterodimers at positions
364 and 370, and the HC2 does not contain an alteration that
disfavors heterodimers; or (2) the HC2 is an IgG1 HC, the HC1 is an
IgG4 HC, the HC2 comprises alterations that disfavor heterodimers
at positions 364 and 370, and the HC1 does not contain an
alteration that disfavors heterodimers; or
[0089] (f) (1) the HC1 and HC2 are IgG1 HCs, the HC1 comprises
alterations that disfavor heterodimers at positions 364 and 370,
and the HC2 comprises the alteration K409R; or (2) the HC1 and HC2
are IgG1 HCs, the HC2 comprises alterations that disfavor
heterodimers at positions 364 and 370, and the HC1 comprises the
alteration K409R.
[0090] 16. The mixture of item 15, wherein
[0091] (a) (1) the HC1 is an IgG1 HC and comprises alterations
D399R/K and K409D/E, and the HC2 is an IgG4 HC does not contain an
alteration that disfavors heterodimers or (2) the HC2 is an IgG1 HC
and comprises alterations D399R/K and K409D/E, and the HC1 is an
IgG4 HC does not contain an alteration that disfavors
heterodimers;
[0092] (b) the HC1 and HC2 are IgG1 HCs, and one of them comprises
alterations D399R/K and K409D/E and the other comprises alteration
K409R;
[0093] (c) (1) the HC1 is an IgG1 HC that comprises alterations
N390R/K and S400D/E or alterations N390D/E and S400R/K, and the HC2
is an IgG4 HC; or (2) the HC1 is an IgG1 HC, and the HC2 is an IgG4
HC that comprises alterations N390R/K and S400D/E or alterations
N390D/E and S400R/K;
[0094] (d) (1) the HC1 and HC2 are IgG1 HCs, and the HC1 comprises
alterations N390R/K and S400D/E or the alterations N390D/E and
S400R/K; or (2) the HC1 and HC2 are IgG1 HCs, and the HC2 comprises
the alterations N390R/K and S400D/E or the alterations N390D/E and
S400R/K;
[0095] (e) (1) the HC1 is an IgG1 HC, the HC2 is an IgG4 HC, the
HC1 comprises the alterations S364K/R and K370D/E, and the HC2 does
not contain an alteration that disfavors heterodimers; or (2) the
HC2 is an IgG1 HC, the HC1 is an IgG4 HC, the HC2 comprises
alterations S364K/R and K370D/E, and the HC1 does not contain an
alteration that disfavors heterodimers; or
[0096] (f) (1) the HC1 and HC2 are IgG1 HCs, the HC1 comprises
alterations S364K/R and K370D/E, and the HC2 comprises alteration
K409R; or (2) the HC1 and HC2 are IgG1 HCs, the HC2 comprises
alterations S364K/R and K370D/E, and the HC1 comprises alteration
K409R.
[0097] 17. The mixture of any one of items 1 to 16, wherein if the
HC1 and/or HC2 is an IgG4 HC, then the IgG4 HC(s) comprise(s) the
alteration S228P.
[0098] 18. The mixture of any one of items 1 to 17, wherein the in
vivo half lives of the first and second antibodies differ by at
least one week.
[0099] 19. The mixture of item 18, wherein the in vivo half lives
of the first and second antibodies differ by at least ten days.
[0100] 20. The mixture of item 19, wherein the in vivo half lives
of the first and second antibodies differ by at least two
weeks.
[0101] 21. The mixture of any one of items 18 to 20, wherein the
antibody with the shorter in vivo half life contains the alteration
M252A, M252L, M252S, M252R, R255K or H435R.
[0102] 22. The mixture of any one items 1 to 21, wherein
[0103] the HC1 comprises a CDR1, CDR2, and CDR3 having the amino
acid sequences SEQ ID NOs: 28, 29, and 30, respectively, the LC1
comprises a CDR1, CDR2, and CDR3 having the amino acid sequences
SEQ ID NOs: 25, 26, and 27, respectively, the HC2 comprises a CDR1,
CDR2, and CDR3 having the amino acid sequences SEQ ID NOs: 31, 32,
and 33, and the LC2 comprises a CDR1, CDR2, and CDR3 having the
amino acid sequences SEQ ID NOs: 34, 35, and 36.
[0104] 23. The mixture of item 22, wherein
[0105] the HC1 comprises a heavy chain variable domain (VH) having
the amino acid sequence of amino acids 1-120 of SEQ ID NO:20
comprising the alterations 44D and 105R, the LC1 comprises a light
chain variable domain (VL) having the amino acid sequence of amino
acids 1-107 of SEQ ID NO:19 comprising the alterations 43D, and
100R, the HC2 comprises a VH having the amino acid sequence of
amino acid 1-119 of SEQ ID NO:21 comprising the alterations 44R and
105D, and the LC2 comprises a VL having the amino acid sequence of
amino acid 1-107 of SEQ ID NO:22 comprising the alterations 43R and
100D.
[0106] 24. The mixture of any one items 1 to 23, wherein
[0107] (a) one of the first and second antibodies binds to human
CTLA4 and the other binds to human PD1,
[0108] (b) both the first and second antibodies bind to human HER2,
but they do not compete for binding to HER2,
[0109] (c) one of the first and second antibodies binds to human
LAG3 and the other binds to human PD1,
[0110] (d) one of the first and second antibodies binds to human
GITR and the other binds to human PD1,
[0111] (d) one of the first and second antibodies binds to human
VEGF and the other binds to human PD1,
[0112] (f) one of the first and second antibodies binds to human
CSFR1a and the other binds to human PD1,
[0113] (g) one of the first and second antibodies binds to human
OX40 and the other binds to human PD1,
[0114] (h) one of the first and second antibodies binds to human
TIGIT and the other binds to human PD1,
[0115] (i) one of the first and second antibodies binds to human
CTLA4 and the other binds to human PDL1,
[0116] (j) one of the first and second antibodies binds to human
VEGF and the other binds to human PDL1,
[0117] (k) one of the first and second antibodies binds to human
OX40 and the other binds to human PDL1,
[0118] (l) one of the first and second antibodies binds to human
CSFR1a and the other binds to human PDL1,
[0119] (m) one of the first and second antibodies binds to human
TIGIT and the other binds to human PDL1,
[0120] (n) one of the first and second antibodies binds to human
Tim3 and the other binds to human PDL1,
[0121] (o) one of the first and second antibodies binds to human
CTLA4 and the other binds to human VEGF,
[0122] (p) one of the first and second antibodies binds to human
CTLA4 and the other binds to human 41BB,
[0123] (q) one of the first and second antibodies binds to human
CD20 and the other binds to human CD37,
[0124] (r) one of the first and second antibodies binds to human
ANG2 and the other binds to human VEGF,
[0125] (s) one of the first and second antibodies binds to human
TNF and the other binds to human IL17a,
[0126] (t) one of the first and second antibodies binds to human
CD38 and the other binds to human CD138,
[0127] (u) one of the first and second antibodies binds to human
EGFR and the other binds to human HER2,
[0128] (v) one of the first and second antibodies binds to human
EGFR and the other binds to human HER3,
[0129] (w) one of the first and second antibodies binds to human
MET and the other binds to human VEGF
[0130] (x) one of the first and second antibodies binds to human
MET and the other binds to human EGFR,
[0131] (y) one of the first and second antibodies binds to human
TSLP and the other binds to human IL33, or
[0132] (z) one of the first and second antibodies binds to human
IL4 and the other binds to human IL13, or
[0133] (aa) one of the first and second antibodies binds to human
PD1 and the other binds to human CD96.
[0134] 25. A full-length antibody comprising two primate and/or
humanized LCs having the same amino acid sequence and two primate
and/or humanized IgG HCs having the same amino acid sequence,
[0135] wherein the HCs each comprise a charged amino acid at
position 181,
[0136] wherein the LCs each comprise a charged amino acid at
position 178, and
[0137] wherein the charge of the amino acid at the HC position 181
is opposite to the charge of the amino acid at the LC position
178.
[0138] 26. A method of making the mixture of antibodies of any one
of items 1 to 24, comprising the steps of:
[0139] (a) culturing the host cell line expressing the antibody
species in a culture medium, and
[0140] (b) recovering the mixture comprising the antibody species
from the culture medium.
[0141] 27. The method of item 26, wherein the host cell line is a
mammalian cell line.
[0142] 28. The method of item 27, wherein the host cell line is a
CHO cell line.
[0143] 29. The method of any one of items 26 to 28, further
comprising a step of purifying the mixture from other components
present in the culture medium.
[0144] 30. A host cell line that produces the mixture of antibodies
of any one of items 1 to 24.
[0145] 31. The host cell line of item 30, which is a mammalian cell
line.
[0146] 32. The host cell of item 31, which is a CHO cell line.
[0147] 33. A nucleic acid or a mixture of nucleic acids encoding
the antibody or the mixture of antibodies of any one of items 1 to
25.
[0148] 34. One or more vector(s) containing the nucleic acid or
mixture of nucleic acids of item 33.
[0149] 35. The vector(s) of item 34, each of which is a mammalian
expression vector.
[0150] 36. The vector(s) of item 34, each of which is a viral
vector.
[0151] 37. The vector(s) of item 36, each of which is an
adenovirus, an adeno-associated virus (AAV), a retrovirus, a
vaccinia virus, a modified vaccinia virus Ankara (MVA), a herpes
virus, a lentivirus, or a poxvirus vector.
[0152] 38. A method of treating a disease comprising administering
to a patient the mixture of any one of items 1 to 24.
[0153] 39. A method of treating cancer comprising administering to
a patient the mixture of item 24(a).
[0154] 40. A method of treating breast cancer comprising
administering to a patient the mixture of item 24(b).
[0155] 41. The method of item 40, wherein the mixture comprises
three major antibody species.
[0156] 42. The method of item 41, wherein the mixture comprises at
most three major antibody species.
[0157] 43. A method of treating a patient having a tumor comprising
injecting into the tumor the mixture of any one of items 1 to
24.
[0158] 44. A method of treating a patient having a tumor comprising
administering directly to the tumor the nucleic acid(s) of item 33
or the vector(s) of any one of items 34 to 37.
[0159] 45. The method of item 44, wherein the nucleic acid(s) or
the vector(s) are injected into the tumor.
[0160] 46. A mixture of antibodies comprising two major antibody
species, which include
[0161] (a) a first antibody comprising a first heavy chain (HC1)
and a first light chain (LC1) and
[0162] (b) a second antibody comprising a second heavy chain (HC2)
and a second light chain (LC2),
[0163] wherein the first and second antibodies are full-length
primate and/or humanized IgG antibodies,
[0164] wherein the first antibody comprises two chains of the HC1
having the same first amino acid sequence and two chains of the LC1
having the same second amino sequence,
[0165] wherein the second antibody comprises two chains of the HC2
having the same third amino acid sequence and two chains of the LC2
having the same fourth amino sequence,
[0166] wherein the HC1 and the HC2 have different amino acid
sequences,
[0167] wherein the LC1 and the LC2 have different amino acid
sequences,
[0168] wherein the mixture comprises not more than ten different
major species of full-length IgG antibodies, and
[0169] wherein the mixture has been produced by a host cell line
into which DNA(s) encoding the HC1, HC2, LC1, and LC2 has (have)
been introduced.
[0170] 47. The mixture of item 46, wherein the mixture comprises
not more than three different major species of full-length
antibodies.
[0171] 48. The mixture of item 47, wherein the DNA(s) encoding the
HC1, HC2, LC1, and LC2 was (were) introduced at the same time.
[0172] 49. The mixture of item 47, wherein
[0173] the DNA(s) encoding the HC1 and LC1 was (were) introduced
before the DNA(s) encoding the HC2 and LC2, or
[0174] the DNA(s) encoding the HC2 and LC2 was (were) introduced
before the DNA(s) encoding the HC1 and LC1.
[0175] 50. The mixture of any one of items 46 to 49, wherein the
HC1 and HC2 are human and/or humanized IgG heavy chains (HCs), and
the LC1 and LC2 are human and/or humanized light chains (LCs).
[0176] 51. The mixture of any one of items 46 to 50, wherein:
[0177] (a) both the first and second antibodies comprise one or
more partner-directing alteration;
[0178] (b) both the first and second antibodies comprise one or
more partner-directing alteration, and at least one of the first
and second antibodies comprises at least one alteration that
disfavors heterodimers; or
[0179] (c) either the first antibody comprises one or more
partner-directing alteration and the second does not or vice
versa.
[0180] 52. The mixture of item 51(c), wherein either the first
antibody comprises one or more alteration that disfavors
heterodimers and the second antibody does not, or vice versa.
[0181] 53. The mixture of item 52, wherein either
[0182] (1) the first antibody comprises one or more
partner-directing alteration and one or more alteration that
disfavors heterodimers and the second antibody does not comprise a
partner-directing alteration or an alteration that disfavors
heterodimers, or
[0183] (2) the second antibody comprises one or more
partner-directing alteration and one or more alteration that
disfavors heterodimers and the first antibody does not comprise a
partner-directing alteration or an alteration that disfavors
heterodimers.
[0184] 54. The mixture of any one of items 46 to 51, which
comprises three major antibody species.
[0185] 55. The mixture of any one of items 46 to 51, which
comprises not more than two major antibody species.
[0186] 56. The mixture of item 55, wherein the HC1 and/or the HC2
contain(s) one or more alteration that disfavors heterodimers.
[0187] 57. The mixture of any one of items 46 to 56, wherein
[0188] (a) (1) the HC1 is an IgG1 or IgG2 heavy chain (HC), the HC2
is an IgG4 HC, the HC1 comprises alterations that disfavor
heterodimers at positions 399 and 409, and the HC2 does not contain
an alteration that disfavors heterodimers; or (2) the HC2 is an
IgG1 or IgG2 HC, the HC1 is an IgG4 HC, the HC2 comprises
alterations that disfavor heterodimers at positions 399 and 409,
and the HC1 does not comprise an alteration that disfavors
heterodimers; or
[0189] (b) (1) the HC1 and HC2 are each an IgG1 or an IgG2 HC, the
HC1 comprises alterations that disfavor heterodimers at positions
399 and 409, and the HC2 comprises the alteration K409R; or (2) the
HC1 and HC2 are each an IgG1 or an IgG2 HC, the HC2 comprises
alterations that disfavor heterodimers at positions 399 and 409,
and the HC1 comprises the alteration K409R.
[0190] 58. The mixture of item 57, wherein
[0191] (a) (1) the HC1 is an IgG1 or IgG2 HC and comprises the
alterations D399R/K and K409D/E, and the HC2 is an IgG4 HC and does
not comprise an alteration that disfavors heterodimers or (2) the
HC2 is an IgG1 or IgG2 HC and comprises the alterations D399R/K and
K409D/E, and the HC1 is an IgG4 HC and does not comprise an
alteration that disfavors heterodimers; or
[0192] (b) the HC1 and HC2 are each an IgG1 or an IgG2 HC, and one
of them comprises the alterations D399R/K and K409D/E and the other
comprises the alteration K409R.
[0193] 59. The mixture of any one of items 46 to 51 and 54 to 56,
wherein the first antibody comprises at least one alteration that
disfavors heterodimers and the second antibody does not contain an
alteration that disfavors heterodimers, or vice versa.
[0194] 60. The mixture of any one of items 46 to 59, wherein
[0195] (a) the first antibody comprises at least one
partner-directing alteration and the second antibody does not;
and
[0196] (b) (1) the first antibody is a human and/or humanized IgG1
antibody, the cysteine at position 220 in the HC1 is substituted
with another amino acid, and the cysteine at position 214 in the
LC1 is substituted with another amino acid, or (2) the first
antibody is a human and/or humanized IgG2 or IgG4 antibody, the
cysteine at position 131 in the HC1 is substituted with another
amino acid, and the cysteine at position 214 in the LC1 is
substituted with another amino acid.
[0197] 61. The mixture of item 60, wherein
[0198] the first antibody is an IgG1 antibody, and it comprises the
alterations C220G/A in the HC1 and C214S/A/G in the LC1; or
[0199] the first antibody is an IgG2 or IgG4 antibody, and it
comprises the alterations C131S/NG in the HC1 and C214S/A/G in the
LC1.
[0200] 62. The mixture of item 61, wherein
[0201] the first antibody is an IgG1 antibody, and it comprises the
alterations C220G in the HC1 and C214S in the LC1; or
[0202] the first antibody is an IgG2 or IgG4 antibody, it comprises
the alterations C131S in the HC1 and C214S in the LC1.
[0203] 63. The mixture of any of items 60 to 62,
[0204] wherein the first antibody is an IgG1 antibody;
[0205] wherein the first antibody comprises at least one pair of
cysteine substitutions at a first pair of amino acid positions;
[0206] wherein the first pair of amino acid positions comprises a
first HC1 position and a first LC1 position; and
[0207] wherein the first HC1 and LC1 positions, respectively, are
selected from the group consisting of: positions 126 and 124;
positions 128 and 118; positions 133 and 117; positions 133 and
209; positions 134 and 116; positions 168 and 174; positions 170
and 162; positions 170 and 176; positions 173 and 160; and
positions 183 and 176.
[0208] 64. The mixture of item 63,
[0209] wherein the first HC1 and LC1 positions, respectively, are
selected from the group consisting of: positions 168 and 174;
positions 173 and 160; and positions 170 and 162.
[0210] 65. The mixture of item 64, wherein
[0211] the first antibody comprises a second pair of cysteine
substitutions at a second pair of positions comprising a second HC1
and second LC1 position, which are different from the first HC1 and
first LC1 positions, respectively, and
[0212] the second HC1 and LC1 positions, respectively, are selected
from the group consisting of: positions 126 and 124; positions 128
and 118; positions 133 and 117; positions 133 and 209; positions
134 and 116; positions 168 and 174; positions 170 and 162;
positions 170 and 176; positions 173 and 160; and positions 183 and
176.
[0213] 66. The mixture of item 65, wherein the first HC1 and LC1
positions are, respectively, 173 and 160, and the second HC1 and
LC1 positions are, respectively, 170 and 162.
[0214] 67. The mixture of any one of items 60 to 62,
[0215] wherein the first antibody is an IgG4 antibody;
[0216] wherein the first antibody comprises at least one pair of
cysteine substitutions at a first pair of amino acid positions;
[0217] wherein the first pair of amino acid positions comprises a
first HC1 position and a first LC1 position; and
[0218] wherein the first HC1 and LC1 positions, respectively, are
selected from the group consisting of: positions 126 and 121;
positions 126 and 124; positions 127 and 121; positions 128 and
118; positions 168 and 174; positions 170 and 162; and positions
173 and 162.
[0219] 68. The mixture of item 67, wherein
[0220] the first antibody comprises a second pair of cysteine
substitutions at a second HC1 position and a second LC1 position,
which are different from the first HC1 and LC1 positions,
respectively, and
[0221] the second HC1 and LC1 positions, respectively, are selected
from the group consisting of: positions 126 and 121; positions 126
and 124; positions 127 and 121; positions 128 and 118; positions
168 and 174; positions 170 and 162; and positions 173 and 162.
[0222] 69. The mixture of any one of items 60 to 62,
[0223] wherein the first antibody is an IgG2 antibody;
[0224] wherein the first antibody comprises at least one pair of
cysteine substitutions at a first pair of contacting amino acid
positions, wherein one position is in the HC1 and the other is in
the LC1.
[0225] 70. The mixture of item 69, wherein the first pair of
contacting amino acid positions in the HC1 and LC1, respectively,
is selected from the group consisting of: 170 and 162; and 173 and
160.
[0226] 71. The mixture of item 69 or 70, wherein the first antibody
comprises a second pair of cysteine substitutions at a second pair
of contacting amino acid positions, wherein one position in the
second pair of contacting amino acid positions is an HC1 position
and the other is an LC1 position, and wherein the HC1 and LC1
positions in the second pair of contacting amino acid positions are
each different from the HC1 and LC1 positions, respectively, in the
first pair of contacting amino acid positions.
[0227] 72. The mixture of item 71, wherein the second pair of
contacting amino acid positions in the HC1 and LC1, respectively,
is selected from the group consisting of: 170 and 162; and 173 and
160.
[0228] 73. The mixture of any one of items 46 to 72, wherein the
first antibody comprises at least one partner-directing alteration
in which a charged amino acid is substituted for another amino
acid.
[0229] 74. The mixture of item 73,
[0230] wherein the first antibody is an IgG1 antibody,
[0231] wherein the first antibody comprises a charge pair of amino
acids,
[0232] wherein one amino acid of the charge pair is in the HC1, and
one amino acid of the charge pair is in the LC1,
[0233] wherein at least one of the amino acids of the charge pair
results from the partner-directing alteration; and
[0234] wherein the charge pair consists of one of the following
amino acid pairs at the following positions in the HC1 and LC1,
respectively: 147D/E and 124K/R; 147D/E and 129K/R; 147D/E and
131K/R; 147D/E and 180K/R; 168D/E and 164K/R; 168D/E and 167K/R; or
168D/E and 174K/R.
[0235] 75. The mixture of item 73,
[0236] wherein the first antibody is an IgG4 antibody,
[0237] wherein the first antibody comprises a charge pair of amino
acids,
[0238] wherein one amino acid of the charge pair is in the HC1, and
one amino acid of the charge pair is in the LC1,
[0239] wherein at least one of the amino acids of the charge pair
results from the partner-directing alteration; and
[0240] wherein the charge pair consists of one of the following
amino acid pairs at the following positions in the HC1 and LC1,
respectively: 133D/E and 117K/R; 137K/R and 114D/E; 137K/R and
116D/E; 147D/E and 124K/R; 147D/E and 129K/R; 147D/E and 131K/R;
147D/E and 178K/R; 147D/E and 180K/R; 168D/E and 164K/R; 168D/E and
167K/R; 168D/E and 173K/R; or 168D/E and 174K/R.
[0241] 76. The mixture of item 73,
[0242] wherein the first antibody is an IgG2 antibody,
[0243] wherein the first antibody comprises a charge pair of amino
acids,
[0244] wherein one amino acid of the charge pair is in the HC1 and
one amino acid of the charge pair is in the LC1,
[0245] wherein at least one of the amino acids of the charge pair
results from the partner-directing alteration.
[0246] 77. The mixture of item 76, wherein the charge pair consists
of 147D/E in the HC1 and 131K/R in the LC1.
[0247] 78. The mixture of any one of items 46 to 59, wherein
[0248] the HC1 and/or the HC2 contain(s) at least one
LC-partner-directing alteration at an HC residue, and
[0249] the LC1 and/or the LC2 contain(s) at least one
HC-partner-directing alteration at an LC residue contacting the HC
residue at which the LC-partner-directing alteration(s) occur(s) in
HC1 and/or HC2, respectively, or contacting a charged amino acid or
cysteine present in the HC1 and/or the HC2, respectively.
[0250] 79. The mixture of item 78, wherein
[0251] the LC partner-directing alteration(s) cause(s) the HC1 and
the HC2 to comprise a charged amino acid at the HC residue in both
the HC1 and the HC2,
[0252] the charged amino acid at the HC residue in the HC1 is
opposite in charge to the charged amino acid at the HC residue in
the HC2,
[0253] the HC-partner-directing alteration(s) cause(s) the LC1 and
the LC2 to comprise a charged amino acid at the LC residue in both
the LC1 and the LC2,
[0254] the charged amino acid at the LC residue in the LC1 is
opposite in charge to the charged amino acid at the LC residue in
the LC2, and
[0255] the charged amino acid at the LC residue in the LC1 is
opposite in charge to the amino acid at the HC residue in the
HC1.
[0256] 80. The mixture of item 78 or 79, wherein the first and
second antibodies comprise one or more of the sets of alterations
selected from the group consisting of:
[0257] (a) the set of alterations wherein positions 44 and 105 in
both the HC1 and the HC2 and 43 and 100 in both the LC1 and the LC2
are each occupied by a charged amino acid, the amino acids at
positions 44 and 105 in the HC1 are each opposite in charge from
those at the same sites in the HC2, the amino acids at positions 43
and 100 in the LC1 are each opposite in charge from those at the
same sites in the LC2, the amino acids at position 44 in the HC1
and the HC2 are opposite in charge to the amino acids at position
100 in the LC1 and the LC2, respectively, and the amino acids at
position 105 in the HC1 and the HC2 are opposite in charge to the
amino acids at position 43 in the LC1 and the LC2,
respectively;
[0258] (b) the set of alterations wherein positions 147 in the HC1
and the HC2 and 131 in the LC1 and the LC2 are each occupied by a
charged amino acid, the amino acid at position 147 in the HC1 is
opposite in charge from that at position 147 in the HC2, the amino
acid at position 131 in the LC1 is opposite in charge from that at
position 131 in the LC2, and the amino acids at position 147 in the
HC1 and the HC2 are opposite in charge to the amino acids at
position 131 in the LC1 and the LC2, respectively;
[0259] (c) the set of alterations wherein positions 168 in the HC1
and the HC2 and 174 in the LC1 and the LC2 are each occupied by a
charged amino acid, the amino acid at position 168 the HC1 is
opposite in charge from that at position 168 in the HC2, the amino
acid at position 174 in the LC1 is opposite in charge from that at
position 174 in the LC2, and the amino acids at position 168 in the
HC1 and the HC2 are opposite in charge to the amino acids at
position 174 in the LC1 and the LC2, respectively; and
[0260] (d) the set of alterations wherein positions 181 in the HC1
and the HC2 and 178 or 180 in the LC1 and the LC2 are each occupied
by a charged amino acid, the amino acid at position 181 in the HC1
is opposite in charge from that at position 181 in the HC2, the
amino acid at position 178 or 180 in the LC1 is opposite in charge
from that at position 178 or 180 in the LC2, and the amino acids at
position 181 in the HC1 and the HC2 are opposite in charge to the
amino acids at position 178 or 180 in the LC1 and the LC2,
respectively.
[0261] 81. The mixture of any one of items 78 to 80, wherein the
first and second antibodies comprise one or more of the sets of
alterations selected from the group consisting of:
[0262] (a) the set of alterations wherein the HC1 comprises 44E/D,
the LC1 comprises 100R/K, the HC2 comprises 44R/K, and the LC2
comprises 100D/E; or the HC2 comprises 44E/D, the LC2 comprises
100R/K, the HC1 comprises 44R/K, and the LC1 comprises 100D/E;
[0263] (b) the set of alterations wherein the HC1 comprises 105E/D,
the LC1 comprises 43R/K, the HC2 comprises 105R/K, and the LC2
comprises 43D/E; or the HC2 comprises 105E/D, the LC2 comprises
43R/K, the HC1 comprises 105R/K, and the LC1 comprises 43D/E;
[0264] (c) the set of alterations wherein the HC1 comprises 147R/K,
the LC1 comprises 131E/D, the HC2 comprises 147E/D, and the LC2
comprises 131R/K; or the HC2 comprises 147R/K, the LC2 comprises
131E/D, the HC1 comprises 147E/D, and the LC1 comprises 131R/K;
[0265] (d) the set of alterations wherein the HC1 comprises 168E/D,
the LC1 comprises 174R/K, the HC2 comprises 168R/K, and the LC2
comprises 174D/E; or the HC2 comprises 168E/D, the LC2 comprises
174R/K, the HC1 comprises 168R/K, and the LC1 comprises 174D/E;
and
[0266] (e) the set of alterations wherein the HC1 comprises 181E/D,
the LC1 comprises 178R/K or 180R/K, the HC2 comprises 181R/K, and
the LC2 comprises 178D/E or 180D/E; or the HC2 comprises 181E/D,
the LC2 comprises 178R/K or 180R/K, the HC1 comprises 181R/K, and
the LC1 comprises 178D/E or 180D/E.
[0267] 82. The mixture of item 81, wherein the first and second
antibodies comprise one or more of the sets of alterations selected
from the group consisting of:
[0268] (a) the set of alterations wherein the HC1 comprises 44D and
105R, the LC1 comprises 43D and 100R, the HC2 comprises 44R and
105D, and the LC2 comprises 43R and 100D; or the HC2 comprises 44D
and 105R, the LC2 comprises 43D and 100R, the HC1 comprises 44R and
105D, and the LC1 comprises 43R and 100D;
[0269] (b) the set of alterations wherein the HC1 comprises 147R,
the LC1 comprises 131D, the HC2 comprises 147D, and the LC2
comprises 131R; or the HC2 comprises 147R, the LC2 comprises 131D,
the HC1 comprises 147D, and the LC1 comprises 131R;
[0270] (c) the set of alterations wherein the HC1 comprises 168D,
the LC1 comprises 174R, the HC2 comprises 168R, and the LC2
comprises 174D; or the HC2 comprises 168D, the LC2 comprises 174R,
the HC1 comprises 168R, and the LC1 comprises 174D; and
[0271] (d) the set of alterations wherein the HC1 comprises 181D,
the LC1 comprises 178R or 180R, the HC2 comprises 181R, and the LC2
comprises 178D or 180D; or the HC2 comprises 181D, the LC2
comprises 178R or 180R, the HC1 comprises 181R, and the LC1
comprises 178D or 178D.
[0272] 83. The mixture of item 82, wherein the first and second
antibodies comprise one or more of the sets of alterations selected
from the group consisting of:
[0273] (a) the set of alterations wherein the HC1 comprises G44D
and Q105R, the LC1 comprises A/V43D and Q/G100R, the HC2 comprises
G44R and Q105D, and the LC2 comprises AA/43R and Q/G100D; or the
HC2 comprises G44D and Q105R, the LC2 comprises A/V43D and Q/G100R,
the HC1 comprises G44R and Q105D, and the LC1 comprises A/V43R and
Q/G100D.
[0274] (b) the set of alterations wherein the HC1 comprises K147R,
the LC1 comprises S131D, the HC2 comprises K147D, and the LC2
comprises S131R; or the HC2 comprises K147R, the LC2 comprises
S131D, the HC1 comprises K147D, and the LC1 comprises S131R;
[0275] (c) the set of alterations wherein the HC1 comprises H168D,
the LC1 comprises S174R, the HC2 comprises H168R, and the LC2
comprises S174D; or the HC2 comprises H168D, the LC2 comprises
S174R, the HC1 comprises H168R, and the LC1 comprises S174D;
and
[0276] (d) the set of alterations wherein the HC1 comprises S181D,
the LC1 comprises T/Y178R or T/S180R, the HC2 comprises S181R, and
the LC2 comprises T/Y178D or T/S180D; or the HC2 comprises S181D,
the LC2 comprises T/Y178R or T/S180R, the HC1 comprises S181R, and
the LC1 comprises T/Y178D or T/S180D.
[0277] 84. The mixture of any one of items 78 to 83,
[0278] wherein the first and/or the second antibody comprise one or
more pairs of cysteine substitutions at one or more pairs of
positions;
[0279] wherein each pair of positions comprises one HC position and
one LC position;
[0280] wherein the HC and LC positions, respectively, in the pairs
of positions are selected from the group consisting of: 126 and
121; 126 and 124; 127 and 121; 128 and 118; 133 and 117; 133 and
209; 134 and 116; 141 and 116; 168 and 174; 170 and 162; 170 and
176; 173 and 160; 173 and 162; and 183 and 176; and
[0281] wherein the first and second antibodies do not comprise
cysteine substitutions at the same pairs of positions.
[0282] 85. The mixture of item 84, wherein the first and second
antibodies comprise one or more of the sets of alterations selected
from the group consisting of:
[0283] (1) positions 170 and 183 in the HC1 and the HC2,
respectively, and 162 and 176 in the LC1 and the LC2, respectively,
are each substituted with cysteine; or positions 170 and 183 in the
HC2 and the HC1, respectively, and 162 and 176 in the LC2 and the
LC1, respectively, are each substituted with cysteine;
[0284] (2) positions 173 and 170 in the HC1 and the HC2,
respectively, and 160 and 162 in the LC1 and the LC2, respectively,
are each substituted with cysteine; or positions 173 and 170 in the
HC2 and the HC1, respectively, and 160 and 162 in the LC2 and the
LC1, respectively, are each substituted with cysteine;
[0285] (3) positions 173 and 183 in the HC1 and the HC2,
respectively, and 160 and 176 in the LC1 and the LC2, respectively,
are each substituted with cysteine; or positions 173 and 183 in the
HC2 and the HC1, respectively, and 160 and 176 in the LC2 and the
LC1, respectively, are each substituted with cysteine;
[0286] (4) positions 170 in the HC1 and the HC2 and 162 and 176 in
the LC1 and the LC2, respectively, are each substituted with
cysteine; or positions 170 in the HC2 and the HC1 and 162 and 176
in the LC2 and the LC1, respectively, are each substituted with
cysteine;
[0287] (5) positions 170 and 173 in the HC1 and the HC2,
respectively, and 162 and 160 in the LC1 and the LC2, respectively,
are each substituted with cysteine; or positions 170 and 173 in the
HC2 and the HC1, respectively, and 162 and 160 in the LC2 and the
LC1, respectively, are each substituted with cysteine;
[0288] (6) positions 173 and 170 in the HC1 and the HC2,
respectively, and 162 and 176 in the LC1 and the LC2, respectively,
are each substituted with cysteine; or positions 173 and 170 in the
HC2 and the HC1, respectively, and 162 and 176 in the LC2 and the
LC1, respectively, are each substituted with cysteine;
[0289] (7) positions 173 in the HC1 and the HC2 and 162 and 160 in
the LC1 and the LC2, respectively, are each substituted with
cysteine; or positions 173 in the HC2 and the HC1 and 162 and 160
in the LC2 and the LC1, respectively, are each substituted with
cysteine;
[0290] (8) positions 183 and 173 in the HC1 and the HC2,
respectively, and 176 and 160 in the LC1 and the LC2, respectively,
are each substituted with cysteine; or positions 183 and 173 in the
HC2 and the HC1, respectively, and 176 and 160 in the LC2 and the
LC1, respectively, are each substituted with cysteine;
[0291] (9) positions 170 and 173 in the HC1 and the HC2,
respectively, and 176 and 160 in the LC1 and the LC2, respectively,
are each substituted with cysteine; or positions 170 and 173 in the
HC2 and the HC1, respectively, and 176 and 160 in the LC2 and the
LC1, respectively, are each substituted with cysteine;
[0292] (10) positions 170 and 183 in the HC1 and the HC2,
respectively, and 176 in the LC1 and the LC2 are each substituted
with cysteine; or positions 170 and 183 in the HC2 and the HC1,
respectively and 176 in the LC2 and the LC1 are each substituted
with cysteine; and
[0293] (11) positions 173 and 170 in the HC1 and the HC2,
respectively, and 162 in both the LC1 and the LC2 are each
substituted with cysteine; or positions 173 and 170 in the HC2 and
the HC1, respectively, and 162 in both the LC2 and the LC1 are each
substituted with cysteine.
[0294] 86. The mixture of item 85, wherein the first and second
antibodies comprise one or more of the sets of alterations selected
from the group consisting of:
[0295] (1) the HC1 comprises F170C, the LC1 comprises S162C, the
HC2 comprises S183C, and the LC2 comprises S176C; or the HC2
comprises F170C, the LC2 comprises S162C, the HC1 comprises S183C,
and the LC1 comprises S176C;
[0296] (2) the HC1 comprises V173C, the LC1 comprises Q160C, the
HC2 comprises F170C, and the LC2 comprises S162C; or the HC2
comprises V173C, the LC2 comprises Q160C, the HC1 comprises F170C,
and the LC1 comprises S162C;
[0297] (3) the HC1 comprises V173C, the LC1 comprises Q160C, the
HC2 comprises S183C, and the LC2 comprises S176C; or the HC2
comprises V173C, the LC2 comprises Q160C, the HC1 comprises S183C,
and the LC1 comprises S176C;
[0298] (4) the HC1 comprises F170C, the LC1 comprises S162C, the
HC2 comprises F170C, and the LC2 comprises S176C; or the HC2
comprises F170C, the LC2 comprises S162C, the HC1 comprises F170C,
and the LC1 comprises S176C;
[0299] (5) the HC1 comprises F170C, the LC1 comprises S162C, the
HC2 comprises V173C, and the LC2 comprises 0160C; or the HC2
comprises F170C, the LC2 comprises S162C, the HC1 comprises V173C,
and the LC1 comprises 0160C;
[0300] (6) the HC1 comprises V173C, the LC1 comprises S162C, the
HC2 comprises F170C, and the LC2 comprises S176C; or the HC2
comprises V173C, the LC2 comprises S162C, the HC1 comprises F170C,
and the LC1 comprises S176C;
[0301] (7) the HC1 comprises V173C, the LC1 comprises S162C, the
HC2 comprises V173C, and the LC2 comprises 0160C; or the HC2
comprises V173C, the LC2 comprises S162C, the HC1 comprises V173C,
and the LC1 comprises 0160C;
[0302] (8) the HC1 comprises S183C, the LC1 comprises S176C, the
HC2 comprises V173C, and the LC2 comprises 0160C; or the HC2
comprises S183C, the LC2 comprises S176C, the HC1 comprises V173C,
and the LC1 comprises Q/E160C;
[0303] (9) the HC1 comprises F170C, the LC1 comprises S176C, the
HC2 comprises V173C, and the LC2 comprises 0160C; or the HC2
comprises F170C, the LC2 comprises S176C, the HC1 comprises V173C,
and the LC1 comprises 0160C;
[0304] (10) the HC1 comprises F170C, the LC1 comprises S176C, the
HC2 comprises S183C, and the LC2 comprises S176C; or the HC2
comprises F170C, the LC2 comprises S176C, the HC1 comprises S183C,
and the LC1 comprises S176C; and
[0305] (11) the HC1 comprises V173C, the LC1 comprises S162C, the
HC2 comprises F170C, and the LC2 comprises S162C; or the HC2
comprises V173C, the LC2 comprises S162C, the HC1 comprises F170C,
and the LC1 comprises S162C.
[0306] 87. The mixture of any one of items 46 to 86, wherein if the
HC1 and/or the HC2 is an IgG4 HC, then the IgG4 HC(s) comprise(s)
228P.
[0307] 88. The mixture of any one of items 46 to 87, wherein the in
vivo half lives of the first and second antibodies differ by at
least one week.
[0308] 89. The mixture of item 88, wherein the in vivo half lives
of the first and second antibodies differ by at least ten days.
[0309] 90. The mixture of item 89, wherein the in vivo half lives
of the first and second antibodies differ by at least two
weeks.
[0310] 91. The mixture of any one of items 88 to 90, wherein the
antibody with the shorter in vivo half life comprises at least one
of the following alterations: M252A, M252L, M252S, M252R, R255K or
H435R.
[0311] 92. The mixture of any one items 46 to 59 and 78 to 91,
wherein
[0312] the HC1 comprises a CDR1, CDR2, and CDR3 having the amino
acid sequences SEQ ID NOs: 28, 29, and 30, respectively, the LC1
comprises a CDR1, CDR2, and CDR3 having the amino acid sequences
SEQ ID NOs: 25, 26, and 27, respectively, the HC2 comprises a CDR1,
CDR2, and CDR3 having the amino acid sequences SEQ ID NOs: 31, 32,
and 33, and the LC2 comprises a CDR1, CDR2, and CDR3 having the
amino acid sequences SEQ ID NOs: 34, 35, and 36.
[0313] 93. The mixture of any one items 46 to 91, wherein
[0314] (a) one of the first and second antibodies binds to human
CTLA4 and the other binds to human PD1,
[0315] (b) both the first and second antibodies bind to human HER2,
but they do not compete for binding to HER2,
[0316] (c) one of the first and second antibodies binds to human
LAG3 and the other binds to human PD1,
[0317] (d) one of the first and second antibodies binds to human
GITR and the other binds to human PD1,
[0318] (d) one of the first and second antibodies binds to human
VEGF and the other binds to human PD1,
[0319] (f) one of the first and second antibodies binds to human
CSFR1a and the other binds to human PD1,
[0320] (g) one of the first and second antibodies binds to human
OX40 and the other binds to human PD1,
[0321] (h) one of the first and second antibodies binds to human
TIGIT and the other binds to human PD1,
[0322] (i) one of the first and second antibodies binds to human
CTLA4 and the other binds to human PDL1,
[0323] (j) one of the first and second antibodies binds to human
VEGF and the other binds to human PDL1,
[0324] (k) one of the first and second antibodies binds to human
OX40 and the other binds to human PDL1,
[0325] (l) one of the first and second antibodies binds to human
CSFR1a and the other binds to human PDL1,
[0326] (m) one of the first and second antibodies binds to human
TIGIT and the other binds to human PDL1,
[0327] (n) one of the first and second antibodies binds to human
Tim3 and the other binds to human PDL1,
[0328] (o) one of the first and second antibodies binds to human
CTLA4 and the other binds to human VEGF,
[0329] (p) one of the first and second antibodies binds to human
CTLA4 and the other binds to human 41BB,
[0330] (q) one of the first and second antibodies binds to human
CD20 and the other binds to human CD37,
[0331] (r) one of the first and second antibodies binds to human
ANG2 and the other binds to human VEGF,
[0332] (s) one of the first and second antibodies binds to human
TNF and the other binds to human IL17a,
[0333] (t) one of the first and second antibodies binds to human
CD38 and the other binds to human CD138,
[0334] (u) one of the first and second antibodies binds to human
EGFR and the other binds to human HER2,
[0335] (v) one of the first and second antibodies binds to human
EGFR and the other binds to human HER3,
[0336] (w) one of the first and second antibodies binds to human
MET and the other binds to human VEGF
[0337] (x) one of the first and second antibodies binds to human
MET and the other binds to human EGFR,
[0338] (y) one of the first and second antibodies binds to human
TSLP and the other binds to human IL33,
[0339] (z) one of the first and second antibodies binds to human
IL4 and the other binds to human IL13,
[0340] (aa) one of the first and second antibodies binds to human
PD1 and the other binds to human CD96,
[0341] (bb) one of the first and second antibodies binds to human
PD1 and the other binds to human SIRP-alpha, or
[0342] (cc) one of the first and second antibodies binds to human
PD1 and the other binds to human CCR8.
[0343] 94. An antibody comprising a primate or humanized IgG CH1
domain and a primate or humanized CL domain,
[0344] wherein the CH1 and CL domains comprise a charge pair of
amino acids,
[0345] wherein one amino acid of the charge pair is in the CH1
domain and the other is in the CL domain, and
[0346] wherein [0347] (1) the CH1 domain is an IgG1 CH1 domain, and
the HC and LC positions, respectively, of the amino acids of the
charge pair are selected from the group consisting of: 181 and 178;
and 168 and 174; or [0348] (2) the CH1 domain is an IgG4 CH1
domain, and the HC and LC positions, respectively, of the amino
acids of the charge pair are selected from the group consisting of:
181 and 180; and 168 and 174.
[0349] 95. An antibody comprising a primate or humanized IgG1 CH1
domain and a primate or humanized kappa CL domain,
[0350] wherein the CH1 and CL domains comprise a pair of contacting
cysteine residues,
[0351] wherein one cysteine of the pair is in the CH1 domain and
the other is in the CL domain, and
[0352] wherein the CH1 and CL positions, respectively, of the pair
of cysteines are selected from the group consisting of: (1) 168 and
174; (2) 133 and 117; (3) 173 and 160; (4) 170 and 162; (5) 170 and
176; (6) 126 and 124; (7) 128 and 118; and (8) 134 and 116.
[0353] 96. The antibody of item 95, wherein the antibody comprises
substitutions of the cysteines at positions 220 in the CH1 domain
and 214 in the CL domain with other amino acids.
[0354] 97. An antibody comprising a primate or humanized IgG4 CH1
domain and a primate or humanized kappa CL domain,
[0355] wherein the CH1 and CL domains comprise a pair of contacting
cysteine residues,
[0356] wherein one of the pair of contacting cysteine residues is
at a CH1 residue and the other is at a CL residue, and
[0357] wherein the CH1 and CL residues, respectively, of the pair
of contacting cysteine residues are selected from the group
consisting of: (1) 168 and 174; (2) 173 and 162; (3) 170 and 162;
(4) 126 and 124; (5) 127 and 121; and (6) 128 and 118.
[0358] 98. The antibody of item 97, wherein the antibody comprises
substitutions of the cysteines at positions 131 in the CH1 domain
and 214 in the CL domain with other amino acids.
[0359] 99. An antibody comprising a primate or humanized IgG2 CH1
domain and a primate or humanized CL domain,
[0360] wherein the antibody comprises substitutions of the
cysteines at positions 131 in the CH1 domain and 214 in the CL
domain with other amino acids, and
[0361] wherein the CH1 and CL domains each comprise a cysteine
substitution, which creates a pair of contacting cysteine
residues.
[0362] 100. The antibody of item 99, wherein the pair of contacting
cysteine residues are selected from the group of positions in the
CH1 and CL domains, respectively, consisting of: (1) positions 173
and 162; and (2) positions 170 and 162.
[0363] 101. The antibody of item 99 or 100, wherein the CH1 and CL
domains each comprise a second cysteine substitution, which creates
a second, different pair of contacting cysteine residues.
[0364] 102. The antibody of item 101, wherein the second pair of
contacting cysteine residues are selected from the group of
positions in the CH1 and CL domains, respectively, consisting of:
(1) positions 173 and 162; and (2) positions 170 and 162.
[0365] 103. The antibody of any one of items 94 to 102, wherein the
antibody is a full-length human or humanized IgG antibody.
[0366] 104. A method of making the mixture of antibodies of any one
of items 46 to 93, comprising the steps of:
(a) culturing the host cell line expressing the mixture of
antibodies in a culture medium, and (b) recovering the mixture of
antibodies from the cell mass or the culture medium.
[0367] 105. The method of item 104, wherein the host cell line is a
mammalian cell line.
[0368] 106. The method of item 105, wherein the host cell line is a
CHO cell line.
[0369] 107. The method of any one of items 104 to 106, further
comprising a step of purifying the mixture of antibodies from other
components present in the cell mass or the culture medium.
[0370] 108. A host cell line that produces the mixture of
antibodies of any one of items 46 to 93.
[0371] 109. The host cell line of item 108, which is a mammalian
cell line.
[0372] 110. The host cell line of item 109, which is a CHO cell
line.
[0373] 111. One or more nucleic acid(s) encoding the antibody or
the mixture of antibodies of any one of items 46 to 103.
[0374] 112. One or more vector(s) containing the nucleic acid(s) of
item 111.
[0375] 113. The vector(s) of item 112, each of which is a mammalian
expression vector.
[0376] 114. The vector(s) of item 112, each of which is a viral
vector.
[0377] 115. The vector(s) of item 114, each of which is an
adenovirus, an adeno-associated virus (AAV), a retrovirus, a
vaccinia virus, a modified vaccinia virus Ankara (MVA), a herpes
virus, a lentivirus, or a poxvirus vector.
[0378] 116. A host cell line containing the nucleic acid(s) and/or
the vector(s) of any one of items 111 to 115.
[0379] 117. A method of treating a disease comprising administering
to a patient having the disease the mixture of antibodies of any
one of items 46 to 93, wherein the disease is a cancer, a metabolic
disease, an infectious disease, or an autoimmune or inflammatory
disease.
[0380] 118. The method of item 117, wherein the disease is a
cancer.
[0381] 119. A method of treating a disease comprising administering
to a patient having the disease an antibody of any one of items 94
to 103.
[0382] 120. A method of treating cancer comprising administering to
a patient the mixture of antibodies of item 93(a).
[0383] 121. A method of treating breast cancer comprising
administering to a patient the mixture of antibodies of item
93(b).
[0384] 122. The method of item 121, wherein the mixture of
antibodies comprises three major antibody species.
[0385] 123. A method of treating a patient having a tumor
comprising injecting into the tumor the antibody or the mixture of
antibodies of any one of items 46 to 103.
[0386] 124. A method of treating a cancer patient comprising
administering to the patient the nucleic acid(s) and/or the
vector(s) of any one of items 111 to 115.
[0387] 125. The method of item 124, wherein the patient has a tumor
and the nucleic acid(s) and/or vector(s) is (are) administered
directly to the tumor.
[0388] 126. The method of item 125, wherein the nucleic acid(s)
and/or the vector(s) are injected into the tumor.
BRIEF DESCRIPTION OF THE FIGURES
[0389] FIG. 1: Alterations used to produce a "MabPair" in a host
cell. The various domains of the antibodies are indicated in the
figure. Antibody 1 is an IgG1, IgG2, or IgG3 antibody in which
residue 409 has been changed to be an arginine or an IgG4 antibody,
which has a naturally occurring arginine at position 409. As
indicated, Antibody 1 binds to Antigen A (indicated by a circle
filled with irregularly-spaced squares). Antibody 2 is an IgG
antibody in which residue 409 has been altered to be aspartic acid
or glutamic acid (K409D/E, indicated by a circled "-") and residue
399 has been altered to be an arginine or lysine (D399R/K,
indicated by a circled "+"). As indicated, Antibody 2 binds to
Antigen B (indicated by a circle filled with a checkerboard
pattern). As indicated by circled "+" and "-" signs, pairs of
charged residues in the VL and VH domains and in the CL and CH1
domains of Antibody 1 and Antibody 2 drive the light chains of
Antibodies 1 and 2 to pair with their cognate heavy chains. These
charged residues may be present in the original amino acid sequence
of the antibody or can be introduced by substituting a charged
amino acid for the amino acid present in the original sequence.
Moreover, these charged residues create a repulsive force between
the heavy chain of Antibody 1 and the light chain of Antibody 2,
and between the heavy chain of Antibody 2 and the light chain of
Antibody 1. Cysteine residues, which can be introduced by
substitution at different locations within the CH1/CL interface of
Antibodies 1 and 2, are indicated by two .COPYRGT.'s joined by a
solid line. Additional disulfide bridges normally present in the
hinge domains of the antibodies are indicated by solid horizontal
lines.
[0390] FIG. 2: Alterations used to produce three different
antibodies (a "3-in-1 mixture") in one host cell. Markings are as
in FIG. 1. Antibody 1 (at left) is an IgG1 antibody that binds to
Antigen A, Antibody 2 is an IgG1 antibody that binds to Antigen B,
and Antibody 3 is a bispecific antibody that binds to both Antigens
A and B, as indicated. Unlike the embodiment shown in FIG. 1,
alterations in the CH3 domain to disfavor heterodimer formation are
not present in Antibody 2.
[0391] FIG. 3: Alternate strategy used to produce a MabPair (Panel
A) or a 3-in-1 mixture (Panel B) of antibodies in one host cell.
Markings are as indicated in FIG. 1. Panel A. Antibody 1 is an
IgG1, IgG2, or IgG3 antibody, in which residue 409 has been changed
to be an arginine, or an IgG4 antibody, which has a naturally
occurring arginine at position 409. As indicated, Antibody 1 binds
to Antigen A. Antibody 1 does not have any alterations in its Fab
region, i.e., the VH-CH1 and VL-CL domains. The naturally occurring
disulfide bridge between the HC and LC is indicated by a heavy line
between the CH1 domain and the carboxy terminus of the LC. Antibody
2 is an IgG antibody in which residue 399 has been altered to be an
arginine or lysine (D399R/K, indicated by a circled "+") and
residue 409 has been altered to be aspartic acid or glutamic acid
(K409D/E, indicated by a circled "-"). As indicated, Antibody 2
binds to Antigen B. Due to amino acid substitutions Antibody 2
lacks the naturally occurring interchain disulfide bond between the
LC and the HC, but has two newly-introduced disulfide bonds between
the CL and CH1 domains (indicated by two .COPYRGT.'s joined by a
solid line). Further, an introduced charge pair (e.g., S131K/R in
CL and K147D/E in CH1 or S174K/R in CL and H168D/E in CH1) is
indicated by circled "+" and "-". Panel B. Markings are as in panel
A. Note that the alterations in the CH3 domain shown in panel A are
absent. Antibody 3 is a bispecific antibody comprising an HC and LC
from each of Antibody 1 and Antibody 2.
[0392] FIG. 4: Antibody species potentially produced by a host cell
transfected with DNA encoding two full-length antibodies. The
circle at left represents a host cell. The longer solid, bent line
and shorter solid line represent the HC and LC of a first antibody,
and the longer dashed, bent line and shorter dashed line represent
the HC and LC of a second antibody. These polypeptides are encoded
by DNAs that have been introduced into the host cell. Outside the
circle at right are shown all the potential antibody species that
could be produced if promiscuous pairings of the various chains are
not prevented by some mechanism. The three species encircled by a
dotted-line rectangle are the species that contain only cognate
HC/LC pairs. All other species include at least one non-cognate
HC/LC pair.
[0393] FIG. 5: Analysis of antibody species formed in the presence
of cysteine substitutions at contacting residues in the HC and the
LC. Experiments are described in Example 2. Briefly, DNAs encoding
an HC with or without alterations and a kappa LC (kLC) with or
without alterations were introduced into a host cell, and the
antibodies produced by the host cell were analyzed by Western
blotting. The designation "aCTLA4(IgG1)KE" used below refers to the
HC of an IgG1 anti-CTLA4 antibody with the alterations C220S (which
eliminates the naturally occurring HC/LC disulfide bridge), D399K,
and K409E. The designation "aPD1(IgG4)" used below refers to the HC
of an IgG4 anti-PD1 antibody that comprises the alterations C131S
(which eliminates the naturally occurring HC/LC disulfide bridge)
and S228P (which prevents IgG4 Fab arm exchange). The designations
"aCTLA4-kLC" and "aPD1-kLC" used below refer to the cognate light
chains of these antibodies, and both comprise the alteration C214S
(which eliminates the naturally occurring HC/LC disulfide bridge).
Other alterations, e.g., (H168C), in these polypeptides or no
further alterations (WT) are indicated in parenthesis following
this description of the polypeptide. Lanes 1, 13, 23, 24, and 34
contain size markers that are not visible in this image. Other
lanes contain samples from transfectants containing DNAs encoding
the following: lane 2, aCTLA4(IgG1)KE(H168C) and aCTLA4-kLC(S174C);
lane 3, aCTLA4(IgG1)KE(K133C) and aCTLA4-kLC(I117C); lane 4,
aCTLA4(IgG1)KE(K133C) and aCTLA4-kLC(F209C); lane 5,
aCTLA4(IgG1)KE(V173C) and aCTLA4-kLC(Q160C); lane 6,
aCTLA4(IgG1)KE(F170C) and aCTLA4-kLC(S162C); lane 7,
aCTLA4(IgG1)KE(F170C) and aCTLA4-kLC(S176C); lane 8,
aCTLA4(IgG1)KE(S183C) and aCTLA4-kLC(S176C); lane 9,
aPD1(IgG4)(H168C) and aPD1-kLC(S174C); lane 10, aPD1(IgG4)(V173C)
and aPD1-kLC(S162C); lane 11, aPD1(IgG4)(F170C) and
aPD1-kLC(S162C); lane 12, aPD1(IgG4)(S183C) and aPD1-kLC(S176C);
lane 14, aCTLA4(IgG1)KE(H168C) and aPD1-kLC(WT); lane 15,
aCTLA4(IgG1)KE(K133C) and aPD1-kLC(WT); lane 16,
aCTLA4(IgG1)KE(V173C) and aPD1-kLC(WT); lane 17,
aCTLA4(IgG1)KE(F170C) and aPD1-kLC(WT); lane 18,
aCTLA4(IgG1)KE(S183C) and aPD1-kLC(WT); lane 19, aPD1(IgG4)(H168C)
and aCTLA4-kLC(WT); lane 20, aPD1(IgG4)(V173C) and aCTLA4-kLC(WT);
lane 21, aPD1(IgG4)(F170C) and aCTLA4-kLC(WT); lane 22,
aPD1(IgG4)(S183C) and aCTLA4-kLC(WT); lane 25, aPD1(IgG4)(WT) and
aCTLA4-kLC(S174C); lane 26, aPD1(IgG4)(WT) and aCTLA4-kLC(I117C);
lane 27, aPD1(IgG4)(WT) and aCTLA4-kLC(F209C); lane 28,
aPD1(IgG4)(WT) and aCTLA4-kLC(Q160C); lane 29 aPD1(IgG4)(WT) and
aCTLA4-kLC(S162C); lane 30, aPD1(IgG4)(WT) and aCTLA4-kLC(S176C);
lane 31, aCTLA4(IgG1)KE(WT) and aPD1-kLC(S174C); lane 32,
aCTLA4(IgG1)KE(WT) and aPD1-kLC(S162C); and lane 33
aCTLA4(IgG1)KE(WT) and aPD1-kLC(S176C). Arrows at left point to
various bands as follows: top arrow, HC-LC/HC-LC, full-length
antibody; second arrow, HC/HC-LC, a three quarters antibody
containing two HCs and one LC; third arrow, HC/HC, an antibody
species containing only two HCs; and bottom arrow, HC-LC, a half
antibody containing one HC and one LC.
[0394] FIG. 6: Analysis of antibody species formed in the presence
of cysteine substitutions at contacting residues in the HC and the
LC. Experiments are described in Example 2. Lanes 1-6 contain cell
supernatants from cells co-transfected with DNAs encoding the HC
and LC of the IgG4 anti-PD1 antibody described in Example 2. All
the anti-PD1 HCs comprise S228P. Other alterations in these
antibodies are as follows: lane 1, wild type (wt) (HC) and (LC);
lane 2, C131S (HC) and C214S (LC); lane 3, C131S plus F126C (HC)
and C214S a plus S121C (LC); lane 4, C131S plus F126C (HC) and
C214S plus Q124C (LC); lane 5, C131S plus P127C (HC) and C214S plus
S121C (LC); and lane 6, C131S plus L128C (HC) and C214S plus F118C
(LC). Lanes 7-11 contain cell supernatants from cells
co-transfected with DNAs encoding the wild wt or altered HC and LC
of the IgG1 anti-CTLA4 antibody described in Example 2. The
alterations in these antibodies are as follows: lane 7, wt (HC) and
(LC); lane 8, C220S (HC) and C214S (LC); lane 9, C220S plus F126C
(HC) and C214S plus Q124C (LC); lane 10, C220S plus L128C (HC) and
C214S plus F118C (LC); and lane 11, C220S plus S134C (HC) and C214S
plus F116C (LC). Lane 12 contains a cell supernatant from a mock
transfection in which no DNA was used. Lanes 13 and 14 contain cell
supernatants from transfections using the IgG1 anti-HER2 antibody
4D5-8 (comprising the amino acid sequences SEQ ID NOs. 19 and 20).
Lanes 15 and 16 contain cell supernatants from transfections using
the IgG1 anti-HER2 antibody 2C4 (which contains the amino acid
sequences of SEQ ID NOs. 21 and 22). The positions of size (in
kilodaltons (kDa)) markers are indicated on the left size of the
image of the Western blot.
[0395] FIG. 7: Chain drop-out transient transfections to assess
HC/LC pairing. Experiments are described in Example 3. Each set of
five samples comes from transfections using various combinations of
the DNAs encoding a first heavy chain (HC1) and a first light chain
(LC1), which together encode a first antibody (an anti-PD1
antibody), and a second heavy chain (HC2) and a second light chain
(LC2), which together encode a second antibody (an anti-CTLA4
antibody). As indicated in the figure, the combinations are as
follows: 1) HC1, LC1, HC2, and LC2; 2) HC1 and LC1; 3) HC1 and LC2;
4) HC2 and LC2; and 5) HC2 and LC1. The designations 18A-18D above
each set of five lanes indicate that the HCs and LCs in these lanes
have the alterations described for the designations 18A-18D in
Table 21. Lanes 21 and 22 contain supernatants from cells
transfected with the full-length anti-HER2 antibodies, 4D5-8
(comprising the amino acid sequences SEQ ID NOs: 19 and 20) and 2C4
(which contains the amino acid sequences of SEQ ID NOs: 21 and 22),
respectively, included as a control to monitor transfection
efficiency.
[0396] FIG. 8: Chain drop-out transient transfections to assess
HC/LC pairing. Experiments are described in Example 3. As indicated
under lanes 1-20, for each pair of variants a set of five DNA
combinations (as described above for FIG. 7) was transfected into
the host cells. The designations 14A-14D above each set of five
lanes indicate that the HCs and LCs in these lanes have the
alterations described for these designations in Table 20. DNAs
encoding anti-HER2 antibodies 4D5-8 and 2C4 were transfected in
duplicate at the same time to monitor transfection efficiency, and
cell supernatants from these transfectants were analyzed in lanes
labeled 5-8 (4D5-8) and 4 (2C4).
[0397] FIG. 9: Chain drop-out transient transfections to assess
HC/LC pairing. Experiments are described in Example 3. As indicated
under lanes 1-20, for each pair of variants a set of five DNA
combinations (as described above for FIG. 7) was transfected into
the host cells. The designations 23A-23D above each set of five
lanes indicate that the HCs and LCs analyzed in these lanes have
the alterations described for these designations in Table 21. DNAs
encoding an anti-PD1 antibody and anti-CTLA4 antibody were
transfected at the same time to monitor transfection efficiency,
and cell supernatants from these tranfectants were analyzed in
lanes labeled a-PD1 and a-CTLA4.
[0398] FIG. 10: Chain drop-out transient transfections to assess
LC/HC pairing in the presence of cysteine substitutions at
contacting residues. Experiments are described in Example 3. Lanes
21-23 in panels B and C contain cell supernatants from control
transfections containing no DNA (lane 21), IgG1 anti-HER2 antibody
4D5-8 (comprising the amino acid sequences SEQ ID NOs. 19 and 20;
lane 22), and IgG1 anti-HER2 antibody 2C4 (which contains the amino
acid sequences of SEQ ID NOs. 21 and 22; lane 23). As indicated,
all other samples are in groups of five and contain cell
supernatants from transfections containing the DNAs encoding
various combinations of HC1, LC1, HC2, and LC2, as explained in
Example 3 and the description of FIG. 7. HC1 and LC1, together,
make up the IgG4 anti-PD1 antibody described in Example 2
comprising no alterations other than S228P (HC1). HC2 and LC2,
together, make up the IgG1 anti-CTLA4 antibody described in Example
2, which comprises only the alterations D399K and K409R (HC). These
antibodies are either comprise no further alterations (designated
"WT") or are altered in various ways in different lanes as
indicated in the tables below. Positions of molecular weight
standards are indicated at left.
TABLE-US-00001 TABLE 1 Alterations in antibody chains in samples
shown in Panel A Anti-PD1 human IgG4 (S228P)* Anti-CTLA4 human IgG1
(D399K, K409E)* Lane HCI LCI HC2 LC2 1 WT WT WT WT 2 WT WT 3 WT WT
4 WT WT 5 WT WT 6 WT WT C220S C214S 7 WT WT 8 WT C214S 9 C220S
C214S 10 WT C220S 11 WT WT C220S, H168C, V173C C214S, Q160C, S174C
12 WT WT 13 WT C214S, Q160C, S174C 14 C220S, H168C, V173C C214S,
Q160C, S174C 15 WT C220S, H168C, V173C 16 WT WT C220S, H168C, F170C
C214S, S162C, S174C 17 WT WT 18 WT C214S, S162C, S174C 19 C220S,
H168C, F170C C214S, S162C, S174C 20 WT C220S, H168C, F170C 21 WT WT
C220S, F170C, V173C C214S, Q160C, S162C 22 WT WT 23 WT C214S,
Q160C, S162C 24 C220S, F170C, V173C C214S, Q160C, S162C 25 WT
C220S, F170C, V173C *Blank boxes indicate the absence of the chain
listed in the heading above the box.
TABLE-US-00002 TABLE 2 Alterations in antibody chains in samples
shown in Panel B Anti-PD1 human IgG4 (S228P)* Anti-CTLA4 human IgG1
(D399K, K409E)* Lane HCI LCI HC2 LC2 1 WT WT WT WT 2 WT WT 3 WT WT
4 WT WT 5 WT WT 6 WT WT C220S, F170C, V173C C214S, Q160C, S162C 7
WT WT 8 WT C214S, Q160C, S162C 9 C220S, F170C, V173C C214S, Q160C,
S162C 10 WT C220S, F170C, V173C 11 WT WT C220S, F170C, V173C, K147D
C214S, Q160C, S162C, S131K 12 WT WT 13 WT C214S, Q160C, S162C,
S131K 14 C220S, F170C, V173C, K147D C214S, Q160C, S162C, S131K 15
WT C220S, F170C, V173C, K147D 16 WT WT C220S, F170C, V173C, K147D
C214S, Q160C, S162C, S131R 17 WT WT 18 WT C214S, Q160C, S162C,
S131R 19 C220S, F170C, V173C, K147D C214S, Q160C, S162C, S131R 20
WT C220S, F170C, V173C, K147D *Blank boxes indicate the absence of
the chain listed in the heading above the box
TABLE-US-00003 TABLE 3 Alterations in antibody chains in samples
shown in Panel C Anti-PD1 human IgG4 (S228P)* Anti-CTLA4 human IgG1
(D399K, K409E)* Lane HCI LCI HC2 LC2 1 WT WT C220S, F170C, V173C,
H168D C214S, Q160C, S162C, S174K 2 WT WT 3 WT C214S, Q160C, S162C,
S174K 4 C220S, F170C, V173C, H168D C214S, Q160C, S162C, S174K 5 WT
C220S, F170C, V173C, H168D 6 WT WT C220S, F170C, V173C, H168D
C214S, Q160C, S162C, S174R 7 WT WT 8 WT C214S, Q160C, S162C, S174R
9 C220S, F170C, V173C, H168D C214S, Q160C, S162C, S174R 10 WT
C220S, F170C, V173C, H168D 11 WT WT C220S, F170C, V173C, K147D,
H168D C214S, Q160C, S162C, S131K, S174K 12 WT WT 13 WT C214S,
Q160C, S162C, S131K, S174K 14 C220S, F170C, V173C, K147D, H168D
C214S, Q160C, S162C, S131K, S174K 15 WT C220S, F170C, V173C, K147D,
H168D 16 WT WT C220S, F170C, V173C, K147D, H168D C214S, Q160C,
S162C, S131R, S174R 17 WT WT 18 WT C214S, Q160C, S162C, S131R,
S174R 19 C220S, F170C, V173C, K147D, H168D C214S, Q160C, S162C,
S131R, S174R 20 WT C220S, F170C, V173C, K147D, H168D *Blank boxes
indicate the absence of the chain listed in the heading above the
box
[0399] FIG. 11: Heterodimer formation of HCs with alterations at
position 392, 370, and 397. As described in Example 4, host cells
were transfected with DNAs encoding a full-length wild type IgG1 HC
and an LC plus a DNA encoding an IgG1 Fc fragment, with or without
alterations as indicated below. Antibodies from the culture media
of transfectants were subjected to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) under non-reducing conditions and
blotted. Antibodies were detected as described in Example 4.
Positions of HC-LC/HC-LC homodimer ("HC/HC"), HC-LC/Fc heterodimer
("HC/Fc"), and Fc/Fc ("Fc/Fc") homodimer are indicated. Alterations
present in the Fc fragment and the percent HC-LC/Fc heterodimer in
each lane are as follows: 1) K392A, 46.9%; 2) K392C, 49.2%; 3)
K392D, 42.9%; 4) K392E, 44.1%; 5) K392F, 42.7%; 6) K392G, 50.2%; 7)
K392H, 44.0%: 8) K392M, 43.3%; 9) K392N, 50.0%; 10) K392P, 45.9%;
11) K392Q, 48.1%; 12) K392R, 50.3%; 13) K392S, 46.3%; 14) K392T,
56.3%; 15) K392V, 54.0%; 16) K392W, 49.5%; 17) K392Y, 49.6%; 18)
WT, 39.7%; 19) K370A, 47.3%; 20) K370C, 62.3%; 21) K370D, 48.8%;
22) K370E, 45.4%; 23) K370F, 43.2%; 24) K370G, 50.7%; 25) K370H,
55.2%; 26) K3701, 59.1%; 27) K370L, 60.6%; 28) K370M, 55.7%; 29)
K370N, 47.4%; 30) K370P, 59.3%; 31) K370Q, 49.2%; 32) K370T, 54.1%;
33) K370V, 51.9%; 34) K370W, 39.7%; 35) K370Y, 47.3%; 36) V397A,
46.9%; 37) V397C, 53.7%; 38) V397D, 52.2%; 39) V397E, 57.5%; 40)
V397F, 51.4%; 41) V397G, 55.3%; 42) V397H, 55.2%; 43) V3971, 51.4%;
44) V397K, 66.5%; 45) V397L, 48.4%; 46) V397M, 52.1%; 47) V397N,
59.4%; 48) V397P, 56.2%; 49) V397Q, 48.0%; 50) V397R, 61.9%; 51)
V397S, 48.9%; 52) V397T, 51.6%; 53) V397W, 49.5%; and 54) V397Y,
49.1%.
[0400] FIG. 12: Heterodimer formation of HCs with alterations at
position(s) 399 and/or 409. This experiment is described in Example
4. The experiment and the markings on the figure are the same as in
FIG. 11 except that, as indicated in the table below, the various
alterations of the Fc fragment are at position(s) 399 and/or 409,
and the full-length HC is a wild type IgG4 HC.
[0401] FIG. 13: Heterodimer formation of IgG1 or IgG4 HCs with Fc
fragments with or without alterations at positions 399 and/or 409.
This experiment is described in Example 4. Markings are as in FIG.
12. A "no HC" or "no Fc" in the table at right indicates that DNA
encoding either the HC or the Fc, respectively, was not introduced
into the host cells whose antibodies are visualized in that lane.
"Ab1" and "Ab2" are two different antibodies. Ab2 IgG4 and Ab2 IgG1
have the same variable domains in an IgG4 and an IgG1 format,
respectively.
[0402] FIG. 14: Heterodimer formation of IgG1 or IgG4 HCs with Fc
fragments with or without alterations at positions 399 and/or 409.
Experiment is described in Example 4. Markings are as in FIG.
13.
[0403] FIG. 15: SDS-PAGE analysis of purified anti-PD1 anti-CTLA4
MabPair antibody mixtures. This experiment is described in Example
5. Non-reduced (left) and reduced (right) samples were run in 4-15%
CRITERION.TM. TGX STAIN-FREE.TM. Precast SDS-PAGE gel and blotted,
and antibodies were detected as described in Example 5. Sizes of
protein molecular weight standards were run in the leftmost lane,
and sizes in kilodaltons (kDa) are indicated. Lanes 1 and 8 contain
samples expected to include three different antibodies, two
different anti-HER2 antibodies, 4D5-8 and 2C4, and a bispecific
antibody containing one HC and one LC from each of these two
antibodies. Lanes 2 and 9 contain samples from cells transfected
with DNAs encoding an anti-CTLA4 IgG1 antibody. Other lanes contain
samples from transfectants containing DNAs encoding antibody
mixtures described in Table 21 as follows: lanes 3 and 10, mixture
17B; lanes 4 and 11, mixture 17C; lanes 5 and 12, mixture 18B;
lanes 6 and 13, mixture 18C; and lanes 7 and 14, mixture 19C.
[0404] FIG. 16: SDS-PAGE analysis of non-reduced and reduced
samples of antibody mixtures. This experiment is described in
Example 5. Leftmost lane in top and bottom panels contains
molecular weight standards. Each group of five lanes contains
varying concentrations of non-reduced (top panel) or reduced
(bottom panel) samples of the antibody mixture indicated above the
lanes. These antibody mixtures are described in Table 20.
[0405] FIG. 17: Analysis of non-reduced antibody mixtures by low pH
cation exchange (CEX) chromatography. The figure shows tracings
from CEX columns run at low pH as described in Example 5. The
horizontal axis shows minutes after the start of the column run,
and the vertical axis shows absorbance at 214 nanometers (indicated
as "AU," which reflects protein concentration) detected in the
column outflow. As indicated, the upper tracing is from an
anti-CTLA4 antibody, the middle tracing is from an anti-PD1
antibody, and the bottom tracing is from the 18C variant antibody
mixture (described in Table 21), which was produced in host cells
transfected with DNA encoding altered versions of anti-PD1 and
anti-CTLA4 antibodies.
[0406] FIG. 18: Mass spectrometry (MS) analysis of Fab fragments
from the purified anti-HER2/anti-HER2 14D 3-in-1 mixture of
antibodies. The variant 14D mixture is described in Table 20. This
experiment is described in Example 5. The Fab fragments generated
by papain digestion were analyzed using a Waters SYNAPT.TM. G2 MS
system (Waters Corporation, Milford, Mass., USA). Above the most
prominent peaks, the masses (in Daltons) are indicated. As
indicated, the vertical axis shows the intensity of the signal (ion
counts per second), and the horizontal axis shows mass in
kilodaltons.
[0407] FIG. 19: MS analysis of anti-CTLA4, anti-PD1 MabPair
antibody mixture. These experiments are described in Example 6. As
indicated, the x axes show deconvoluted mass, and the y axes show
counts, which are reflective of the quantity of protein at a given
mass. Panel A. This panel shows analysis of the IgG1 anti-CTLA4 111
antibody without alteration. Panel B. This panel shows analysis of
the anti-CTLA4 111 antibody containing the alterations K147D,
F170C, V173C, C220G, R255K, D399R, and K409E in the HC and S131K,
0160C, S162C, and C214S in the LC. Panel C. This panel shows
analysis of a MabPair mixture of an unaltered version of the IgG4
anti-PD1 antibody described in Example 2 and the engineered IgG1
anti-CTLA4 111 antibody analyzed in panel B. Panel D. This panel
shows a higher resolution analysis of the MabPair mixture analyzed
in panel C. As indicated, Area Under Curve (AUC) analysis was
performed to determine the relative amounts of each antibody in
this MabPair.
[0408] FIG. 20: MS analysis of Fab' fragments from an
anti-PD1/anti-CTLA4 MabPair antibody mixture. The experiment is
described in Example 6. Panel A. This panel shows a drawing of an
IgG antibody (at left) and an IgG antibody digested with IdeS
Protease (second from left) and further treated with
2-mercaptoethyl amine (2-MEA) and ethylenediaminetetraacetic acid
(EDTA) (at right). The names of the various fragments generated are
indicated. Panel B. This panel shows MS analysis of the Fab'
fragments generated by IdeS Protease digestion and 2-MEA/EDTA
treatment of the same MabPair that was analyzed in panel D of FIG.
19. As indicated, the x axis shows deconvoluted mass, and the y
axis shows counts, which are reflective of the quantity of protein
at a given mass. Expected masses of the two Fab' fragments
containing cognate HC/LC pairs are indicated, as are the actual
masses of the fragments detected and the calculated error.
[0409] FIG. 21: Liquid chromatography of reduced and non-reduced
lysyl endopeptidase digests of an engineered anti-CTLA4 antibody.
This experiment is described in Example 6. Panel A. This panel
shows the expected amino acid sequences of two of the peptides
resulting from lysyl endopeptidase digestion of the engineered
anti-CTLA4 antibody analyzed in FIG. 19, panel B. See Tables 7 and
11. Expected disulfide bonds are indicated by lines. Panel B. This
panel shows the column profile of a non-reduced sample of the lysyl
endopeptidase digestion of the engineered anti-CTLA4 antibody
described in Example 6. The x axis indicates the time since the
start of the chromatography (expressed as acquisition time in
minutes), and the y axis shows the UV Response, which is reflective
of the quantity of protein in the column effluent. The peak
corresponding to linked chains A and B (see panel A) was identified
by its mass and is indicated by an arrow with the label "3
disulfide-linked peptide." Panel C. This panel shows the column
profile of a reduced sample of the lysyl endopeptidase digestion of
the engineered anti-CTLA4 antibody. X and y axes are the same as in
panel B. The new peaks corresponding to chains A and B (see Panel
A) were identified by their masses and are indicated.
[0410] FIG. 22: Determination of monoisotopic masses of peptides
resulting from lysyl endopeptidase digestion of an engineered
anti-CTLA4 antibody. Experiments are described in Example 6. Panel
A. MS analysis of an expected disulfide-linked peptide from a
non-reduced lysyl endopeptidase digestion of the engineered
anti-CTLA4 antibody (labeled "3 disulfide-linked peptide" in FIG.
21, panel B). The x axis shows mass/charge ratio (m/z ratio), and
the y axis shows counts, which reflect the quantity of ions of a
given mass. Panel B. This panel shows MS analysis of the chain A
peptide (indicated in FIG. 21, panel C) from the reduced lysyl
endopeptidase digestion of the engineered anti-CTLA4 antibody.
Panel C. This panel shows MS analysis of the chain B peptide
(indicated in FIG. 21, panel C) from the reduced lysyl
endopeptidase digestion of the engineered anti-CTLA4 antibody.
[0411] FIG. 23: Mass spectrometry analysis of an IgG2/IgG4 MabPair.
These experiments are described in Example 7. As indicated, the x
axes show deconvoluted mass in atomic mass units (amu), and the y
axes show counts, which are reflective of the quantity of protein
at a given mass. Panel A. This panel shows mass spectrometry
analysis of deglycosylated antibodies produced by cells transfected
with DNAs encoding the engineered IgG2 and unaltered IgG4
antibodies described in Example 7. Panel B. This panel shows mass
spectrometry analysis of the Fab' fragments generated from
antibodies produced by cells transfected with DNAs encoding the
engineered IgG2 and unaltered IgG4 antibodies described in Example
7.
[0412] FIG. 24: Anti-PD1 potency of antibody mixtures. This
experiment is described in Example 8. The samples represented by
the various curves are indicated. The horizontal axis indicates the
log of the anti-PD1 antibody concentration. In the case of the
antibody mixtures, this amount was based on the antibody
concentration determination made in Example 7. The vertical axis
indicates the mean plus or minus the standard error of the mean of
the relative luminescence units (RLU.+-.SEM).
[0413] FIG. 25: Anti-CTLA4 potency of antibody mixtures. This
experiment is described in Example 9. The samples represented by
the various curves are indicated. The horizontal and vertical axes
are designated as in FIG. 16, except that the horizontal axis
represents the log of the anti-CTLA4 antibody concentration rather
than the log of the anti-PD1 antibody concentration.
[0414] FIG. 26: Potency of an anti-CTLA4 antibody in an
anti-CTLA4/anti-PD1 MabPair. Procedures are described in Example
10. The x axis shows the concentration of anti-CTLA4 antibody used
in each assay, and the y axis shows the RLU.+-.SEM. The symbols and
lines identify the samples as indicated in the legend in the upper
left corner of the graph and explained in Example 10. The single
filled circle in the lower right corner represents data from an
unrelated human IgG1 antibody.
[0415] FIG. 27: Effects of anti-HER2 antibody mixtures on breast
cancer cell viability. This experiment is described in Example 11.
The antibodies and antibody mixtures used as samples in the
experiment are indicated as follows. IgG1, a control IgG1/KLC
antibody; 4D5-8, anti-HER antibody 4D5-8; 2C4, anti-HER2 antibody
2C4; 2C4+4D5-8, a mixture of the two anti-HER2 antibodies 4D5-8 and
2C4; and 14D, 15C, 13D, 14C, and 15D, the anti-HER2 antibody
mixtures described in Table 20. The vertical axis indicates the
RLU.+-.SEM. The horizontal axis indicates the concentration of the
antibody or the antibody mixture in the sample in nanomoles/liter
(nM). As indicated, the top and bottom panels contain data from
samples using BT-474 cells and SK-BR-3 cells, respectively.
TABLE-US-00004 [0416] BRIEF DESCRIPTION OF THE SEQUENCE LISTING
Sequence Listing Number Description SEQ ID NO:1 Consensus amino
acid sequence for human VH domains SEQ ID NO:2 Consensus amino acid
sequence for CH1 domains SEQ ID NO:3 Amino acid sequence of a human
IgG1 CH1 domain SEQ ID NO:4 Amino acid sequence of a human IgG2 CH1
domain SEQ ID NO:5 Amino acid sequence of a human IgG3 CH1 domain
SEQ ID NO:6 Amino acid sequence of a human IgG4 CH1 domain SEQ ID
NO:7 Amino acid sequence of a human IgG1 Fc fragment SEQ ID NO:8
Amino acid sequence of a human IgG2 Fc fragment SEQ ID NO:9 Amino
acid sequence of a human IgG3 Fc fragment SEQ ID NO:10 Amino acid
sequence of a human IgG4 Fc fragment SEQ ID NO:11 Consensus amino
acid sequence of a human VL domain SEQ ID NO:12 Consensus amino
acid sequence of a CL.kappa. domain SEQ ID NO:13 Consensus amino
acid sequence of a CL.lamda. domain SEQ ID NO:14 Amino acid
sequence of a human CL.kappa. domain (IMGT accession no. J00241)
SEQ ID NO:15 Amino acid sequence of a human CL.kappa. domain (IMGT
accession no. M11736) SEQ ID NO:16 Amino acid sequence of a human
CL.kappa. domain (IMGT accession no. M11737) SEQ ID NO:17 Amino
acid sequence of a human CL.kappa. domain (IMGT accession no.
AF0017732) SEQ ID NO:18 Amino acid sequence of a human CL.kappa.
domain (IMGT accession no. AF11387) SEQ ID NO:19 Amino acid
sequence of the LC of 4D5-8 SEQ ID NO:20 Amino acid sequence of the
HC of 4D5-8 SEQ ID NO:21 Amino acid sequence of the HC of 2C4 SEQ
ID NO:22 Amino acid sequence of the LC of 2C4 SEQ ID NO:23 Amino
acid sequence of the HC of anti-PD1 antibody 16137 SEQ ID NO:24
Amino acid sequence of the LC of anti-PD1 antibody 16137 SEQ ID
NO:25 Amino acid sequence of the LC CDR1 of the anti-HER2 antibody
4D5-8 SEQ ID NO:26 Amino acid sequence of the LC CDR2 of the
anti-HER2 antibody 4D5-8 SEQ ID NO:27 Amino acid sequence of the LC
CDR3 of the anti-HER2 antibody 4D5-8 SEQ ID NO:28 Amino acid
sequence of the HC CDR1 of the anti-HER2 antibody 4D5-8 SEQ ID
NO:29 Amino acid sequence of the HC CDR2 of the anti-HER2 antibody
4D5-8 SEQ ID NO:30 Amino acid sequence of the HC CDR3 of the
anti-HER2 antibody 4D5-8 SEQ ID NO:31 Amino acid sequence of the HC
CDR1 of the anti-HER2 antibody 2C4 SEQ ID NO:32 Amino acid sequence
of the HC CDR2 of the anti-HER2 antibody 2C4 SEQ ID NO:33 Amino
acid sequence of the HC CDR3 of the anti-HER2 antibody 2C4 SEQ ID
NO:34 Amino acid sequence of the LC CDR1 of the anti-HER2 antibody
2C4 SEQ ID NO:35 Amino acid sequence of the LC CDR2 of the
anti-HER2 antibody 2C4 SEQ ID NO:36 Amino acid sequence of the LC
CDR3 of the anti-HER2 antibody 2C4 SEQ ID NO:37 Nucleotide sequence
encoding the heavy chain of anti-CTLA4 antibody 1E1 SEQ ID NO:38
Amino acid sequence of the heavy chain of the anti-CTLA4 antibody
1E1 SEQ ID NO:39 Nucleotide sequence encoding the light chain of
the anti-CTLA4 antibody 1E1 SEQ ID NO:40 Amino acid sequence of the
light chain of the anti-CTLA4 antibody 1E1 SEQ ID NO:41 Amino acid
sequence of the CH1, hinge, CH2, and CH3 domains of an unaltered
human IgG2 antibody SEQ ID NO:42 Amino acid sequence of the CH1,
hinge, CH2, and CH3 domains of an engineered human IgG2 antibody
SEQ ID NO:43 SEQ ID NO:43: Amino acid sequence of the CL kappa
domain of a human antibody SEQ ID NO:44 SEQ ID NO:43: Amino acid
sequence of the CL kappa domain of an engineered human antibody
REFERENCE TO SEQUENCE LISTING
[0417] This application includes a sequence listing submitted
electronically, in a file entitled SB001WO_ST25.txt, created on
Apr. 21, 2017 and having a size of 64 kilobytes (KB), which is
incorporated by reference herein.
DETAILED DESCRIPTION
[0418] Described herein are mixtures of antibodies and methods for
producing mixtures of antibodies containing a limited number of
major species of antibodies, optionally not more than two, three,
four, five, six, seven, eight, nine, or ten, from host cells,
optionally cells from a single host cell line, that have been
transfected with DNA encoding at least two different antibodies,
optionally full-length primate IgG antibodies, with different
binding specificities. In some embodiments, DNAs encoding at least
two different heavy chains (HCs) and at least two different light
chains (LCs) can be introduced into the host cells. In some
embodiments, the host cells can be transfected with DNAs encoding
at least two, but not more than four, different antibodies with
different binding specificities. In some embodiments, DNAs encoding
at least two, but not more than four, different HCs and at least
two, but not more than four, different LCs can be introduced into
the host cells. In some embodiments, the sequences of all of the
transfected DNAs encoding HCs and LCs can be mutated so that the
amino acid sequences of the antibodies are altered such that
non-cognate HC/LC pairings are disfavored and cognate HC/LC
pairings are highly favored. See FIGS. 1 and 2. Where two different
HCs are introduced into the host cells, one or both of the two
different HCs can, optionally, be altered such that heterodimer
formation is disfavored. In some embodiments, only one heavy chain
is altered to discourage heterodimer formation. In some embodiments
where DNAs encoding only two different antibodies are introduced
into the host cells, only one of the antibodies encoded by the DNAs
comprises one or more partner-directing alterations such that
cognate HC/LC pairing is favored, whereas the other antibody does
not comprise such alterations. See FIG. 3. In such embodiments,
this altered antibody can also comprise one or more alterations
that disfavor heterodimer formation. See FIG. 3. In some
embodiments, one of the antibodies does not comprise alterations
that disfavor heterodimer formation or alterations that favor
cognate HC/LC pairing. See FIG. 3. In some further embodiments,
only one of the two antibodies comprises one or more
partner-directing alteration(s), and only one of the two antibodies
comprises one or more alteration(s) that disfavor heterodimer
formation. In such embodiments, the antibody that comprises the
partner-directing alteration(s) may not comprise the alteration(s)
that disfavor heterodimer formation and vice versa. Alternatively,
one antibody can be unaltered and the other can comprise one or
more partner-directing alteration(s) and one or more alteration(s)
disfavoring heterodimer formation. In some embodiments, only two
major antibody species are produced by the host cell, where each HC
is paired predominantly to its cognate LC, and most of the
antibodies are tetramers containing two heavy chains having the
same amino acid sequence and two light chains having the same amino
acid sequence (referred to herein as "MabPairs"). See FIGS. 1 and
3. In other embodiments, the host cell produces three different
major species of antibodies, two different monospecific antibodies
each comprising two HCs with the same amino acid sequence and two
LCs with the same amino acid sequence plus a functional bispecific
antibody comprising two different HCs and two different LCs
(referred to herein as a "3-in-1 mixture" of antibodies). See FIGS.
2 and 3. Described herein are (1) methods for producing mixtures of
antibodies comprising a limited number of major species in host
cells, optionally in a host cell line, (2) antibodies and mixtures
of antibodies comprising alterations that facilitate cognate HC/LC
pairing and, in some embodiments, alterations that disfavor
formation of heterodimeric HC/HC pairs, (3) host cells and/or host
cell lines transfected with DNA encoding at least two different HCs
and two different LCs altered as described herein, (4) nucleic
acids, e.g., DNAs, encoding such antibodies and mixtures, which may
be carried on one or more vectors, and (5) methods of treatment
using such antibodies, mixtures of antibodies, and/or nucleic acids
encoding them.
Definitions
[0419] A "3-in-1 mixture" of antibodies, as meant herein, refers to
a mixture of antibodies made in a host cell line into which DNAs
encoding two different IgG antibodies has been introduced. A 3-in-1
mixture comprises three different major species of antibodies, that
is, the two different IgG antibodies plus a bispecific antibody
comprising a cognate HC/LC pair from each of the two IgG
antibodies. A 3-in-1 mixture does not refer to mixtures of
antibodies made by separate populations of host cells into which
different DNAs encoding the different antibodies have been
separately introduced.
[0420] An "alteration that disfavors heterodimers," as meant
herein, is a substitution, insertion, or deletion of a single amino
acid within a CH3 domain amino acid sequence in an antibody,
optionally a human, humanized, or primate CH3 domain amino acid
sequence, where the substitution, insertion, or deletion disfavors
the formation of heterodimers in the context of a mixture of
antibodies. An antibody can comprise more than one alteration that
disfavors heterodimers, and multiple alterations that disfavor
heterodimers can occur at multiple sites in one or more antibodies
in a mixture of antibodies. A single alteration that disfavors
heterodimer formation need not be completely effective in
eliminating heterodimers, or effective by itself, to be considered
an "alteration that disfavors heterodimers," as long as it is
partially effective and/or effective when paired with one or more
other alterations. Included among the alterations can be the
substitution of a charged residue for the residue present in the
wild type sequence. Alternatively, a substitution can create a
steric obstacle to proper HC/HC pairing such as a "protuberance"
abutting against another "protuberance." Protuberances or knobs are
described in U.S. Pat. No. 8,679,785, col. 12, line 12 to col. 13,
line 2, which is incorporated herein by reference. Whether one or
more alteration(s) has (have) an effect on HC/HC heterodimer
formation can be determined by the methods described in Example 4.
Data from such experiments is shown in FIGS. 11-14. Alterations
that disfavor heterodimers occur at "domain interface residues."
Domain interface residues are discussed in U.S. Pat. No. 8,592,562
in Table 1 and accompanying text, which are incorporated herein by
reference. Such domain interface residues are said to be
"contacting" residues or are said to "contact" each other if they
are predicted to be physically close, i.e., at most 12 angstroms
(.ANG.) between the alpha carbons (C.alpha., i.e., the carbon
between the amino and the carboxyl moiety of the amino acid) of the
two amino acids or at most 5.5 .ANG. between a side chain heavy
atom (any atom other than hydrogen) of one amino acid and any heavy
atom of the other amino acid according to known structure models.
Such structures are available online, for example, through the
Protein Data Bank (available at
http://www.rcsb.org/pdb/home/home.do) or through the INTERNATIONAL
IMMUNOGENETICS INFORMATION SYSTEM.RTM. (IMGT; available at
http://www.imgt.org). In Table 4 below, examples of contacting
residues at the CH3/CH3 interface in a human IgG antibody are
listed.
TABLE-US-00005 TABLE 4 Contacting residues at a human IgG CH3/CH3
interface Residues in second CH3* having Contacting a heavy atom
within 4.5 angstroms residue in of a side chain heavy atom of the
first CH3* contacting amino acid in first CH3 Q347 K360 Y349 S354,
D356, E357, K360 T350 S354, R355 L351 L351, P352, P353, S354, T366
S354 Y349, T350, L351 R355 T350 D356 Y349, K439 E357 Y349, K370
K360 Q347, Y349 S364 L368, K370 T366 L351, Y407 L368 S364, K409
K370 E357, S364 N390 S400 K392 L398, D399, S400, F405 T394 T394,
V397, F405, Y407 P395 V397 V397 T393, T394, P395 D399 K392, K409
S400 N390, K392 F405 K392, T394, K409 Y407 T366, T394, Y407, S408,
K409 K409 L368, D399, F405, Y407 K439 D356 *Numbering is according
to Edelman et al. (1969), Proc. Natl. Acad. Sci. USA 63: 78-85,
which is incorporated herein in its entirety
[0421] Examples of alterations that disfavor heterodimers include,
e.g., D399K/R plus K/R409D/E in a primate and/or humanized IgG
heavy chain, optionally in the context of a mixture of antibodies
that includes another IgG antibody comprising 409R. As shown in
Table 8 below, human IgG4 antibodies have an arginine (R) at
position 409, while human IgG1, IgG2, and IgG3 antibodies have a
lysine (K) at position 409. In particular instances where an IgG1,
IgG2, or IgG3 antibody comprises the alteration K409R, this
alteration is not considered to be an "alteration that disfavors
heterodimers" (which is an exception to the definition above), as
meant herein, since there is a naturally occurring R at position
409 in human IgG4 HCs.
[0422] An "amino acid," an "amino acid residue," a "residue," or a
"position," within a HC or LC amino acid sequence refers to an
amino acid at a position numbered as shown in Tables 5-11. Thus,
for example, it is possible for two different HC amino acid
sequences to have the same or different amino acids at a particular
position in the two HC amino acid sequences. Further, an "HC
position," an "HC residue," an "LC position," or an "LC residue"
refers to an amino acid at a position in any HC or LC amino acid
sequence numbered as shown in Tables 5-11.
[0423] An "antibody," as meant herein, is a protein that contains
at least one heavy chain variable (VH) domain or light chain
variable (VL) domain. An antibody often contains both VH and VL
domains. VH and VL domains are described in full detail in, e.g.,
Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST,
FIFTH EDITION, U.S. Department of Health and Human Services, Public
Health Service, National Institutes of Health, NIH Publication No.
91-3242, 1991, pp. xvi-xix and pp. 103-533, which are incorporated
by reference herein. "Antibody" includes molecules having different
formats such as single chain Fv antibodies (scFv, which contain VH
and VL regions joined by a linker), Fab, F(ab).sub.2, Fab', scFv:Fc
antibodies (as described in Carayannopoulos and Capra, Ch. 9 in
FUNDAMENTAL IMMUNOLOGY, 3.sup.rd ed., Paul, ed., Raven Press, New
York, 1993, pp. 284-286, which is incorporated herein by
reference), bispecific antibodies and monovalent antibodies in any
of a variety of formats, and full-length and IgG antibodies as
defined below, among other possible formats for an antibody.
[0424] A "bispecific antibody," as meant herein, binds to two
different epitopes, which can reside on one target molecule or on
two separate target molecules. A bispecific antibody can be a
full-length antibody, IgG antibody, or an antibody having a
different format.
[0425] A "bivalent antibody," as meant herein, can simultaneously
bind to two epitopes, which can be identical or different and can
reside on one target molecule or on two separate target
molecules.
[0426] A "charge pair," of amino acids, as meant herein, is a pair
of oppositely charged amino acids at "contacting" amino acid
residues as defined herein. Such charged amino acids can be on the
same polypeptide chain or on different polypeptide chains.
[0427] A "charged" amino acid, as meant herein, is an acidic or
basic amino acid that can have a charge at near-physiologic pH.
These include the acidic amino acids glutamic acid (E) and aspartic
acid (D), which are negatively charged at physiologic pH, and the
basic amino acids arginine (R) and lysine (K), which are positively
charged at physiologic pH. The weakly basic amino acid histidine,
which can be partially charged at near-physiologic pH, is not
within the definition of "charged" amino acid herein. To avoid
confusion, a positive charge is considered to be "opposite" to a
negative charge, as meant herein. Thus, for example, amino acid
residues E and R are opposite in charge.
[0428] A "cognate" HC in the context of a mixture of antibodies, as
meant herein, is the HC that a particular LC is known to pair with
to form a binding site for a particular antigen. For example, if a
known full-length Antibody X binds to Antigen X, the Antibody X HC
is the cognate HC of the Antibody X LC, and vice versa, in the
context of a mixture of antibodies that comprises Antibody X, among
other antibodies. Further, if the mixture also comprises an
Antibody Y, the antibody Y HC is "non-cognate" with respect to the
Antibody X LC and vice versa.
[0429] A "complementarity determining region" (CDR) is a
hypervariable region within a VH or VL domain. Each VH and VL
domain contains three CDRs called CDR1, CDR2, and CDR3. The CDRs
form loops on the surface of the antibody and are primarily
responsible for determining the binding specificity of an antibody.
The CDRs are interspersed between four more conserved framework
regions (called FR1, FR2, FR3, and FR4) as follows:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Positions of CDRs in a VH and a VL
are indicated in Tables 5 and 9, respectively. Kabat et al.
position the VH CDRS as follows: CDR1 is at positions 31-35 (with
possible insertions numbered 35A and 35B); CDR2 is at positions
50-65 (with possible insertions numbered 52A-52C); and CDR3 is at
positions 95-102 (with possible insertions numbered 100A-100K).
Kabat et al., supra, at xvii. Kabat et al. position the VL CDRs as
follows: CDR1 is at positions 24-34 (with possible insertions
numbered 27A-27F); CDR2 is at positions 50-56; and CDR3 is at
positions 89-97 (with possible insertions numbered 95A-95F).
[0430] A "cysteine substitution," as meant herein, refers to an
amino acid substitution in a protein where a cysteine is
substituted for any other amino acid.
[0431] Amino acid alterations within two or more related sequences
"differ," as meant herein, (1) if they occur at different sites
within two amino acid sequences that are the same or within two
amino acid sequences that belong to the same class (e.g., VH
domains) and can be aligned to a common numbering system via
conserved amino acids, and/or (2) if the alteration is different,
e.g., a different amino acid is substituted at the same site within
two amino acid sequences that are otherwise the same or that belong
to the same class or different numbers of amino acids and/or
different amino acids are inserted into or deleted from two amino
acid sequences that are otherwise the same or that belong to the
same class. Of course, amino acid alterations in two or more
unrelated sequences also "differ" from each other. Two or more
antibodies are "different," as meant herein, if the amino acid
sequences of all the polypeptide chains included in the antibody
are not "the same," as meant herein.
[0432] Two or more amino acid sequences are "different," as meant
herein, if they could not be encoded by the same DNA sequence.
Thus, amino acid sequences that differ only because of
post-translational modifications are not "different" as meant
herein.
[0433] An "Fc fragment," as meant herein, comprises most or all of
a hinge domain, plus a CH2 and a CH3 domain from an HC. For
example, amino acid sequences of human IgG Fc fragments are shown
in Table 8.
[0434] A "full-length antibody," as meant herein, comprises (1) two
heavy chains of any isotype each comprising at least a VH domain, a
first heavy chain constant (CH1) domain, a hinge domain, a second
heavy chain constant (CH2) domain, and a third heavy chain constant
(CH3) domain, and (2) two light chains, which can be either kappa
(.kappa.) or lambda (.lamda.) chains, each comprising a VL and a
light chain constant (CL) domain. These domains are described in
detail Kabat et al., supra, pp. xv-xix and 647-699, which pages are
incorporated herein by reference. The numbering system of Kabat et
al., supra, is used for the VH and VL domains (see Tables 5 and 9
below), and the EU system (Edelman et al. (1969), Proc. Natl. Acad.
Sci. USA 63: 78-85, which is incorporated herein in its entirety)
is used for the CL, CH1, hinge, CH2, and CH3 domains. See Tables
6-8, 10, and 11.
[0435] A "heavy chain (HC)," as meant herein, comprises at least
VH, CH1, hinge, CH2, and CH3 domains. An HC including all of these
domains could also be referred to as a "full-length HC." Some
isotypes such as IgA or IgM can contain additional sequences, such
as the IgM CH4 domain. The numbering system of Kabat et al., supra,
is used for the VH domain (see Table 5 below), and the EU system
(Edelman et al. (1969), Proc. Natl. Acad. Sci. USA 63: 78-85, which
is incorporated herein in its entirety) is used for the CH1, hinge,
CH2, and CH3 domains. Tables 5 to 8 below provide a more specific
picture of HC amino acid sequences.
TABLE-US-00006 TABLE 5 Consensus sequence of human VHs 1 2 3 4 5 6
7 8 9 10 11 12 13 14 15 L G P 16 17 18 19 20 21 22 23 24 25 26 27
28 29 30 S V L S C G T L V T 31 32 33 34 35 35A 35B 36 37 38 39 40
41 42 43 W R Q G K Q 44 45 46 47 48 49 50 51 52 52A 52B 52C 53 54
55 G L W 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 R 71 72 73 74
75 76 77 78 79 80 81 82 82A 823 82C S L 83 84 85 86 87 88 89 90 91
92 93 94 95 96 97 D Y C 98 99 100 100A 100B 100C 100D 100E 100F
100G 100H 100I 100J 100K 101 102 103 104 105 106 107 108 109 110
111 112 113 W Q G V V S (SEQ ID NO: 1) Table 5 shows conserved
amino acids based on the human VH amino acid sequences (I-III) in
Kabat et al. (supra). Numbering is according to Kabat et al.,
supra. Site numbers within the CDRs are written in bold italics.
Position numbers with letters after them, e.g., 100A, may or may
not be filled by an amino acid due to the varying lengths of CDRs.
A single boldface amino acid at a particular position indicates an
"invariant" amino acid in all three classes of human VH domains as
described by Kabat et al. (supra). At sites of interest where the
amino acid at a given position is most commonly one amino acid or
either of two amino acids, those amino acids are indicated in plain
text. Site numbers in underlined boldface indicate positions that
are described as being altered herein. Positions where no amino
acid is designated did not meet the criteria stated above.
[0436] Table 5 shows that there are numerous conserved amino acids
that would allow alignment of any VH sequence with the conserved
amino acids spaced as shown above by eye. Alternatively, a novel
sequence could be aligned with a known VH sequence using alignment
software, for example, alignment software available on the
International ImMunoGeneTics (IMGT) Information System.RTM. (for
example, IMGT/DomainGapAlign, which is available at
http://www.imgt.org or CLUSTAL Omega (Sievers et al., (2011),
Molecular Systems Biology 7(1): 539).
[0437] Table 6 below shows a consensus amino acid sequence of CH1
domains.
TABLE-US-00007 TABLE 6 CH1 consensus 118 119 120 121 122 123 124
125 126 127 128 129 130 131 132 P P L 133 134 134 136 137 138 139
140 141 142 143 144 145 146 147 R/K C L K 148 149 150 151 152 153
154 155 156 157 158 159 160 161 162 P W 163 164 165 166 167 168 169
170 171 172 173 174 175 176 177 H F V A 178 179 180 181 182 183 184
185 186 187 188 189 190 191 192 T S S 193 194 195 196 197 198 199
200 201 202 203 204 205 206 207 C 208 209 210 211 212 213 214 215
(SEQ ID NO: 2) TABLE 6: The numbering is the numbering according to
Edelman et al. (supra). The single amino acids shown in boldface
below the numbers are "invariant" residues according to Kabat et
al. (supra) from alignments of CH1 domains from a variety of
species. Sites selected for alteration herein (131, 133, 147, 168,
170, 173, 176, 181, and 183) are shown in underlined boldface. At
these sites, the most common one or two amino acids in the 63
primate CH1 sequences reported in Kabat et al. (supra) are shown in
plain text. Positions where no amino acid is designated were not
"invariant" and were not selected for alteration.
[0438] Table 7 below shows an alignment human CH1 domains of the
IgG1, IgG2, IgG3 and IgG4 isotypes. This alignment highlights the
very strong conservation of sequence among these closely-related
CH1 domains.
TABLE-US-00008 TABLE 7 Alignment of human IgG1, IgG2, IgG3, and
IgG4 CH1 domains 118 120 130 140 150 160 170 177 * * * * * * * *
IgG1 ASTKGPSVFPLAPSS STSGGTAALGCLV DYFPEPVTVSWNSGALTSGV T PA LQ S
IgG2 ASTKGPSVFPLAP S STSESTAALGCLV DYFPEPVTVSWNSGALTSGV T PA LQ S
IgG3 ASTKGPSVFPLAP S STSGGTAALGCLV DYFPEPVTVSWNSGALTSGV T PA LQ S
IgG4 ASTKGPSVFPLAP S STSESTAALGCLV DYFPEPVTVSWNSGALTSGV T PA LQ S
178 180 190 200 210 215 * * * * * * IgG1 GLY L
SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 3) IgG2 GLY L
SVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 4) IgG3 GLY L
SVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV (SEQ ID NO: 5) IgG4 GLY L
SVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 6) Table 7: The amino
acid sequences of representative CH1 domains of human IgG1, IgG2,
IgG3 and IgG4 antibodies were obtained from IMGT web page,
accession numbers J00228, J00230, X03604, and K01316, respectively,
and aligned with CLUSTALW software. Residues are numbered according
to the EU system of Edelman et al., supra. "Invariant" residues
according to Kabat et al., supra are shown in boldface. These
residues are highly conserved, but not completely invariant.
Residues that are underlined and in boldface italics are sites at
which substitutions have been made and tested as reported in the
Examples below.
[0439] Table 8 below shows an alignment of human IgG Fc regions of
the four human IgG subclasses, IgG1, IgG2, IgG3, and IgG4. This
alignment shows the differences between these subclasses, as well
as the high sequence conservation.
TABLE-US-00009 TABLE 8 Amino acid sequences of human IgG Fc regions
IgG1 ----------------------------------------------- IgG2
----------------------------------------------- IgG3
ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP IgG4
----------------------------------------------- 216 226 236 246 256
266 * * * * * * IgG1
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF IgG2
ERKCCVE---CPPCPAPPVA-GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF IgG3
EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF IgG4
ESKYG---PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF 276
286 296 306 316 326 * * * * * * IgG1
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG2
NWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT IgG3
KWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG4
NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT 336
346 356 366 376 386 * * * * * * IgG1
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG2
ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG3
ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTP IgG4
ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP 396
406 416 426 436 446 * * * * * * IgG1
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)
IgG2 PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 8) IgG3 PMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
(SEQ ID NO: 9) IgG4
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:
10)
[0440] A "host cell line" into which DNA(s) encoding one or more
proteins has been introduced refers to a cell line derived from a
single cell following the introduction of the DNA, e.g., by
transfection. Methods for for isolating such clonal cell lines
following the introduction of DNA are well known in the art and
include limiting dilution, among other possible methods that can
include visually determining the existence of only one cell in a
particular sample. See, e.g., Wewetzer (1995), J. Immunol. Methods
179(1): 71-76, Underwood and Bean (1988), J. Immunol. Meth. 107(1):
119-128.
[0441] "Human," nucleotide or amino acid sequences or nucleic acids
or proteins include those that occur naturally in a human. Many
human nucleotide and amino acid sequences are reported in, e.g.,
Kabat et al., supra, which illustrates the use of the word "human"
in the art. A "human" amino acid sequence or protein, as meant
herein, can contain one or more insertions, deletions, or
substitutions relative to a naturally-occurring sequence, with the
proviso that a "human" amino acid sequence or protein does not
contain more than 10 insertions, deletions, and/or substitutions of
a single amino acid per every 100 amino acids. Similarly, a human
nucleic acid (e.g., DNA) or nucleotide sequence does not contain
more than 30 insertions, deletions, and/or substitutions of a
single nucleotide per every 300 nucleotides. In the particular case
of a VH or VL sequence, the CDRs are expected to be extremely
variable, and, for the purpose of determining whether a particular
VH or VL amino acid sequence (or the nucleotide sequence encoding
it) is a "human" sequence, the CDRs (or the nucleotides encoding
them) are not considered part of the sequence.
[0442] A "humanized" nucleotide sequence encoding an antibody or
antibody domain or a "humanized" amino acid sequence of an antibody
or antibody domain, as meant herein, is a sequence that originated
in a non-human organism but was engineered to be as similar as
possible to a human sequence as possible without sacrificing the
desired properties of the antibody, e.g., binding to a certain
antigen with a certain avidity, among many possible desired
properties. The process of humanization generally involves changing
all constant domains to be human constant domains. In the variable
domains, the original CDRs can be used to replace the CDRs of a
human antibody sequence that is as similar as possible to the
original variable domain (a process often referred to as CDR
grafting). However, one or more changes in the framework regions
may also be required. Thus, the amino acid sequence of a humanized
antibody may or may not fall within the definition of "human"
immediately above. This process is described in, e.g., Zhang and
Ho, Scientific Reports 6: 33878; doi: 10.1038/srep33878 (2016) and
Miethe et al. PLOS One; doi: 10.137/journal.pone.0161446 (2016),
both of which are incorporated herein by reference.
[0443] An "IgG antibody," as meant herein, refers to a full-length
antibody, as defined herein, of the IgG isotype, including human,
humanized, and primate antibodies of the IgG1, IgG2, IgG2, and IgG4
isotype subclasses.
[0444] The term "isotype," as meant herein, refers to whether the
heavy chain constant regions in an antibody, i.e., the CH1, hinge,
CH2, and CH3 domains, are of the IgG, IgD, IgM, IgA, or IgE class
or a subclass thereof, such as IgG1, IgG2, IgG3, or IgG4. Such
isotypes are known in the art and are described and explained in
detail in, e.g., Janeway et al., The Immune System in Health and
Disease, 5.sup.th ed., sections 4-15 to 4-19, Garland Science, New
York, 2001 (available at
http://www.ncbi.nlm.nih.gov/books/NBK27106/).
[0445] A "light chain (LC)," as meant herein, comprises a VL domain
and a light chain constant (CL) domain, which can be a kappa
(CL.kappa.) or lambda (CL.lamda.) domain. These domains, including
exemplary amino acid sequences thereof, are described in Kabat et
al., supra, pages xiii-lix, 103-309, and 647-660, which are
incorporated herein by reference. The numbering system used herein
for the light chain is that described in Kabat et al., supra for
the VL domain and that described in Edelman et al., supra for the
CL domain, as illustrated in Tables 9-11 below.
TABLE-US-00010 TABLE 9 Consensus sequence of human VL domains 1 2 3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
27A 27B 27C 27D 27E 27F G C 28 29 30 31 32 33 34 35 36 37 38 39 40
41 42 43 44 W A P S P 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
60 I/V P 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 R F S G S L
76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 A/G Y Y/F 91 92 93 94
95 95A 96 97 98 99 100 101 102 103 104 F G Q/G G T 105 106 106A 107
108 109 (SEQ ID NO: 11) TABLE 9: The numbering is according to
Kabat et al. (supra). Numbers in bold italics indicate the
positions of the CDRs. Position numbers with letters after them,
e.g., 27A, may or may not be filled by an amino acid, due to the
varying lengths of CDRs. Invariant residues for all human light
chains in Kabat et al. (supra) are shown as bold letters indicating
the amino acid found at that position. At selected sites, the one
to three most common amino acids found at that site are indicated
in plain text. In addition, many other amino acids are invariant or
highly conserved within some subgroups of kappa or lambda VL
domains, which can aid in categorizing a particular amino acid
sequence as a VL domain. Sites selected for alteration herein, as
reported in the Examples below, are indicated by boldface
underlined type. Positions where no amino acid is designated and/or
the number is not shown in boldface underlined type do not meet the
criteria stated above.
TABLE-US-00011 TABLE 10 Consensus sequence and numbering for CL
domains 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122
123 .kappa. P I P P .lamda. P L P P 124 125 126 127 128 129 130 131
132 133 134 135 136 137 138 139 .kappa. S V C .lamda. A V C 140 141
142 143 144 145 146 147 148 149 150 151 152 153 154 155 .kappa. P V
W .lamda. P V W 156 157 158 159 160 161 162 163 164 165 166 167 168
169 170 171 .kappa. Q S T .lamda. E T P 172 173 174 175 176 177 178
179 180 181 182 183 184 185 186 187 .kappa. S S S T L T L .lamda.
A/M S S Y L S L 188 189 190 191 192 193 194 195 196 197 198 199 200
201 202 203 .kappa. C H .lamda. C H 204 205 206 207 208 209 210 211
212 213 214 .kappa. F C (SEQ ID NO: 12) .lamda. V C (SEQ ID NO: 13)
TABLE 10: The numbering is according to Edelman et al. (supra),
which is the same as the numbering of Kabat et al. (supra) for CL
domains. The amino acids shown in bold below the numbers are
"invariant" residues according to Kabat et al. (supra) from
alignments of both kappa and lambda CL domains from a variety of
species. As indicated at selected sites (131, 160, 162, 174, 176,
and 178), amino acids conserved in the ten human kappa chains (top)
and 28 human lambda chains (below) reported in Kabat et al. (supra)
are shown in plain text. In cases where either of two different
amino acids are found at one of these sites, the more common amino
acid is shown prior to the less common, e.g., A/M. Bold underlined
numbers indicate sites that were altered as reported in the
Examples below. In addition, many other amino acids are invariant
or highly conserved within some subgroups of CL.kappa. or CL.lamda.
domains, which can aid in categorizing a particular amino acid
sequence as a CL domain. Positions where no amino acid is
designated and/or the number is not shown in boldface underlined
type do not meet the criteria stated above.
TABLE-US-00012 TABLE 11 Alignment of human kappa chain CL domains
108 120 130 140 150 160 167 * * * * * * * J00241 RTVAAPSVF FPPSDE
LKSGTA VVCLLNNFYPREAKVQWKVDNALQSGNS ESV EQD M11736 RTVAAPSVF FPPSDE
LKSGTA VVCLLNNFYPREAKVQWKVDNALQSGNS ESV EQE M11737 RTVAAPSVF FPPSDE
LKSGTA VVCLLNNFYPREAKVQRKVDNALQSGNS ESV EQE AF017732 RTVAAPSVF
FPPSDE LKSGTA VVCLLNNFYPREAKVQWKVDNALQSGNS ESV EQD AF113887
RTVAAPSVF FPPSDE LKSGTA VVCLLNNFYPREAKVQWKVDNALQSGNS ESV EQD 168
170 180 190 200 210 214 * * * * * * * J00241 SKDSTY LSSTL
LSKADYEKHKVYACEVTHQGLSSPVTKS NRGEC (SEQ ID NO: 14) M11736 SKDSTY
LSSTL LSKADYEKHKVYAGEVTHQGLSSPVTKS NRGEC (SEQ ID NO: 15) M11737
SKDSTY LSSTL LSKADYEKHKVYACEVTHQGLSSPVTKS NRGEC (SEQ ID NO: 16)
AF017732 SKDSTY LSSTL LSKADYEKHKLYACEVTHQGLSSPVTKS NRGEC (SEQ ID
NO: 17) AF113887 SKDSTY LSNTL LSKADYEKHKVYACEVTHQGLSSPVTKS NRGEC
(SEQ ID NO: 18) Table 11: The amino acid sequences of human CLk
domains in this table are from the International ImMunoGeneTics
information system .RTM. (IMGT) web page (http://www.imgt.org). The
accession number of each sequence is shown to the left of the
sequence, and the sequences were aligned with CLUSTALW software
(available at http://www.genome.jp/tools/clustalw/). Numbering is
according to Edelman et al. supra. The boldface residues are
invariant residues according to Kabat et al., supra. Invariant
sites where substitutions were made and the resulting antibodies
were tested as reported in the Examples below are indicated by
boldface and underlined amino acids. The bolded, italicized, and
underlined residues are other sites where substitutions were made
and the resulting antibodies were tested as reported in the
Examples below.
[0446] An "LC-partner-directing alteration," as meant herein, is a
substitution, insertion, or deletion of a single amino acid at the
HC/LC interface within a VH or CH1 amino acid sequence, optionally
a substitution of a charged amino acid or a cysteine for the
naturally occurring amino acid, which causes an HC, optionally a
human, humanized, and/or primate IgG HC, containing the altered VH
and/or CH1 amino acid sequence to associate more strongly with an
LC, optionally one containing an "HC-partner-directing alteration"
at a contacting amino acid residue.
[0447] Similarly, an "HC-partner-directing alteration" is the
substitution, insertion, or deletion of a single amino acid in the
HC/LC interface within a VL or CL amino acid sequence, optionally a
substitution of a charged amino acid or a cysteine for the
naturally occurring amino acid, which causes an LC, optionally a
human, humanized, and/or primate kappa or lambda LC, containing the
altered VL or CL amino acid sequence to associate more strongly
with an HC, optionally one containing an LC-partner-directing
alteration at a contacting amino acid residue. In some embodiments,
a contacting pair of HC- and LC-partner-directing alterations can
be substitutions of charged amino acids having opposite charges,
which form a "charge pair," as defined above. In other embodiments,
a charged amino acid already exists at one of the contacting sites
of the HC or LC so that alteration of only one chain is required to
create a charge pair favoring formation of a cognate HC/LC pair. In
other embodiments, cysteine residues can be introduced at
contacting sites so that disulfide bridges between a cognate HC/LC
pair can form. In further embodiments, HC- and LC-partner-directing
alterations can be substitutions or pre-existing amino acids that
create a knob and a hole (or a protuberance and a cavity) at
contacting residues as described in U.S. Pat. No. 8,679,785, the
relevant portions of which are incorporated herein by reference.
The HC can be of the IgG, IgA, IgD, IgM, or IgE isotype, optionally
IgG1, IgG2, IgG3, or IgG4. HC- and LC-partner-directing alterations
occur at contacting amino acid positions that form part of the
HC/LC interface. Interface residues in the CL and CH1 domains
include those within 4.5 .ANG., as explained in U.S. Pat. No.
8,592,562, Tables 4 and 5 and accompanying text in columns 10 and
11, all of which is incorporated herein by reference. Contacting
residues in the CH1 and CL domains are catalogued in Table 12
below.
TABLE-US-00013 TABLE 12 Contacting residues between CH1 and CL CH1
residue CL.kappa. residue CL.lamda. residue 125 123 119 126 121,
123, 124 117, 119, 120 127 121 117, 119 128 118, 133 114, 129 129
118 114 130 118 139 116 140 116 141 116, 118, 135 112, 114 142 118
114 143 114 145 124, 131 127, 129, 173 147 124, 131 125, 127 148
125 168 137, 138, 174 133, 163, 169 169 164 170 135, 162, 164, 174,
176 131, 133, 169, 171 171 162, 164 158, 161, 171 172 158 173 160,
162 156, 158, 173 174 160 156 175 160 156 176 156 181 173 182 173
183 176 129, 131, 173 185 135 114, 131 187 137 213 123 119 218
122
[0448] In the case of contacting residues on the interface between
the VH and VL domains, pairs of residues, one in the VH and one in
the VL domain, suitable for alteration were selected using the
follow criteria: (1) the residues are buried or partially buried,
i.e., inaccessible in the tertiary structure of a full-length
antibody, (2) the residues are spatially close, that is, where the
Ca's of the two amino acids are within about 12 .ANG., or where
there is at most 5.5 .ANG. between a side chain heavy atom (any
atom other than hydrogen) of one amino acid and any heavy atom of
the other amino acid according to known structure models, (3) the
residues are highly conserved, although they need not be totally
invariant, and (4) the residues are not within or interacting with
the complementarity determining regions (CDRs). Examples of such
contacting residues include, without limitation, the following:
position 44 (VH) and position 100 (VL); position 39 (VH) and
position 38 (VL); and position 105 (VH) and position 43 (VL). A
change in the strength of HC/LC association due to HC- and/or
LC-partner-directing alterations can be measured by determining the
relative amounts of various antibody species in a host cell into
which DNA encoding at least two different antibodies has been
introduced. As explained in detail in Examples 5 and 6 and shown in
FIGS. 18, 20, and 23, panel B, the size differences between Fab
fragments arising from antibodies having different HC/LC pairings
can usually be distinguished by mass spectrometry (MS). Use of MS
to identify various antibody species is discussed in, e.g.,
Thompson et al. (2014), mAbs 6:1, 197-203, which is incorporated
herein in its entirety. To quickly screen many different variants,
chain drop out experiments as described in Example 3 and shown in
FIGS. 7-10 were performed, particularly in cases where expected Fab
fragments are essentially identical in size or where expected Fab
fragments do not appear in MS results and thus may be uniquely
susceptible to papain. Examples of contacting pairs of LC- and
HC-partner-directing alterations include, without limitation, the
following: K147D/E in an HC and S131R in an LC; and H168D/E in an
HC and S174R in an LC. As these examples illustrate "contacting"
pairs of LC- and HC-partner directing alterations can include amino
acids opposite in charge. Many other examples are disclosed in the
Description and Examples below. Alternatively, LC- and
HC-partner-directing alterations could be "protuberance in cavity"
style alterations as described in U.S. Pat. No. 8,679,785. The
portions of this patent describing these kinds of alterations,
especially col. 12, line 12 to col. 14, line 5, are incorporated
herein by reference. The term "partner-directing alteration" refers
to HC- and/or LC-partner-directing alterations.
[0449] A "MabPair," as meant herein, is a mixture of antibodies
comprising two, and not more than two, major species of antibodies.
A MabPair can be made in a host cell line (as defined above) into
which DNA encoding two different IgG antibodies, i.e., two
different heavy chains and two different light chains, has been
introduced. A MabPair can also be made in a cell population into
which DNA(s) encoding two different IgG antibodies has (have) been
introduced, where a clonal host cell line is not purified from the
cells into which the DNA(s) was (were) introduced. An example of
this kind of situation could involve transiently transfecting
DNA(s) encoding two different IgG antibodies into, e.g., 293 or
ExpiCHO cells, and subsequently obtaining the antibodies produced
by the cells from the cell supernatant of the transfected cells.
Mixtures of two antibodies that are made from more than one host
cell line are not MabPairs as meant herein. Further, mixtures of
two antibodies made from two separate populations of cells, where
DNA encoding one antibody has been introduced into one cell
population and DNA encoding the other antibody has been introduced
into the other cell population, are also not MabPairs as meant
herein.
[0450] A "major species" of antibody in the context of a mixture of
antibodies, as meant herein, is a particular antibody species that
makes up at least 10% of the total amount of antibodies within the
mixture. To determine how many major species are in a mixture of
antibodies, low pH CEX chromatography as described in Example 5 and
shown in FIG. 17 can be performed. The percentage of the total
amount of antibody that each species in an antibody mixture
comprises can be determined using the areas under the peaks of
absorbance in the column outflow.
[0451] A "minor species" of antibody within a mixture of
antibodies, as meant herein, comprises less than 10% of the total
amount of antibodies in an antibody mixture. This can be determined
by low pH CEX chromatography as described in the definition of
"major species."
[0452] A "primate," nucleotide or amino acid sequence or nucleic
acid or protein includes molecules and sequences that occur
naturally in a primate. Primates include animals from a number of
families including, without limitation, prosimians (including
lemurs), new world monkeys, chimpanzees, humans, gorillas,
orangutans, gibbons, and old world monkeys. Specific primate
species include, without limitation, Homo sapiens, Macaca mulata
(rhesus macaque), Macaca fascicularis (cynomolgus monkey), and Pan
troglodytes (chimpanzee), among many others. Many primate
nucleotide and amino acid sequences are known in the art, e.g.,
those reported in, e.g., Kabat et al., supra. Generally, "primate"
amino acid sequence, as meant herein, can contain one or more
insertions, deletions, or substitutions relative to a
naturally-occurring primate sequence, with the proviso that a
"primate" amino acid sequence does not contain more than 10
insertions, deletions, and/or substitutions of a single amino acid
per every 100 amino acids. Similarly, a primate nucleotide sequence
can contain insertions, deletions, or substitutions relative to a
naturally-occurring primate sequence, but does not contain more
than 30 insertions, deletions, and/or substitutions of a single
nucleotide per every 300 nucleotides. In the particular case of a
VH or VL sequence, the CDRs are expected to be extremely variable,
and, for the purpose of determining whether a particular VH or VL
amino acid sequence (or the nucleotide sequence encoding it) is a
"primate" sequence, the CDRs (or the nucleotides encoding them) are
not considered part of the sequence.
[0453] Two amino acid sequences are "the same," as meant herein, if
the two sequences could be encoded by the same DNA sequence. That
is, amino acid sequences that differ only as a result of
post-translational modifications, e.g., elimination of a
carboxyl-terminal lysine or cyclization of N-terminal glutamate or
glutamine residues, are "the same" as meant herein.
[0454] A "target molecule," as meant herein, is a molecule to which
an antibody, e.g., an antibody in a mixture described herein,
specifically binds. In some embodiments, a target molecule is a
"target protein," i.e., a protein to which an antibody specifically
binds.
Mixtures of Antibodies and Methods of Producing them
[0455] Described herein are mixtures of antibodies having different
binding specificities that are produced in host cells into which
DNA(s) encoding the antibodies has (have) been introduced. The
antibodies can be human, humanized, and/or primate full-length IgG
antibodies. Also described are methods for producing such mixtures.
The number of different major species of antibodies in the mixtures
can be limited, e.g., not more than 2, 3, 4, 5, 6, 7, 8, 9, or 10
major species. The HCs and/or LCs of one or more of the antibodies
can comprise LC- and/or HC-partner-directing alterations. In some
embodiments, the HCs of one or more of the antibodies can comprise
one or more alterations that disfavor heterodimers. In some
embodiments, one or more major species of antibody in a mixture can
be without such alterations. These alterations can serve to limit
the number of major species of antibodies produced by the host
cells.
[0456] The method for producing the mixtures of antibodies can
comprise introducing DNA encoding the mixtures of antibodies
described herein into host cells, culturing the host cells, and
recovering the mixture of antibodies from the cell mass or culture
medium. DNA encoding the different antibodies in a mixture can be
introduced into host cells at the same time or at different times.
For example, DNA encoding a second antibody can be introduced into
a host cell population that already produces a first antibody
encoded by DNA that was previously introduced into the host cell
population. Alternatively, DNA encoding both antibodies can be
introduced into the host cells at the same time. Further, after
introduction of DNAs encoding multiple antibodies into the host
cells, a clonal "host cell line" (as defined above) that produces
the antibodies can be isolated from the population of cells into
which the DNAs were introduced. Alternatively, mixtures of
antibodies can be produced by a host cell population into which the
DNAs were introduced. As explained in detail below, the
alteration(s) in the antibodies can limit the number of major
species of antibodies produced by the host cells. The mixture can
be obtained from a host cell culture supernatant or the cell mass,
can be further purified, and can be formulated as appropriate for
use as a pharmaceutical.
[0457] More specifically, DNAs encoding at least two, three, or
four different antibodies, optionally full-length IgG antibodies,
binding to different epitopes and/or targets can be introduced into
a host cell. The encoded antibodies can each comprise two HCs with
the same amino acid sequence and two LCs with the same amino acid
sequence, and each of the encoded antibodies can have both HCs and
LCs that differ in amino acid sequence from the HCs and LCs of the
other encoded antibody or antibodies. In some embodiments, the
antibodies can be two full-length antibodies, each comprising two
heavy chains having the same amino acid sequence and two light
chains having the same amino acid sequence. Optionally, the
antibodies are primate and/or human and/or humanized IgG
antibodies. In some embodiments, at least one pair of oppositely
charged residues, i.e., charge pairs, or cysteine residues at
contacting sites in a cognate HC/LC pair (where at least one of
these charged residues or cysteines results from an alteration) can
be found in the interface between the LC of each antibody and its
cognate HC. In other embodiments, such charge pairs and/or pairs of
contacting cysteine residues can be found in one or more of the
antibodies in the mixture, but need not be present in all
antibodies in the mixture. Alterations in the LC and HC that create
such pairs are called HC-partner-directing alterations and
LC-partner-directing alterations, respectively. Each antibody can
comprise multiple contacting pairs of LC- and/or
HC-partner-directing alterations or can comprise no pairs of LC-
and/or HC-partner-directing alterations. Optionally, there can be
additional alterations in the CH3 domains that disfavor the
formation of heterodimeric HC/HC pairs. Such alterations can be
present in one or more of the antibodies and can be absent from one
or more antibodies. The host cell population or host cell line can
be cultured, and the mixture of antibodies can be recovered in the
cell mass and/or the culture medium. The mixture of antibodies can
be further purified, and the mixture can be formulated as is
appropriate for its pharmaceutical use.
[0458] In embodiments where DNAs encoding only two full-length
antibodies (Ab1 and Ab2) are introduced into the host cells and
each antibody comprises one or more HC- and/or LC-partner-directing
alteration(s) such that few if any non-cognate HC/LC pairs form,
either two (called herein "MabPair") or three (called herein
"3-in-1 mixture") major species of antibodies can be produced by
the host cells. See FIGS. 1-4. If the HCs can form heterodimeric
HC/HC pairs, a 3-in-1 mixture comprising Ab1, Ab2, and a bispecific
antibody comprising one HC and one LC from each antibody can be
produced by the host cells. If the antibodies do not form
heterodimeric HC/HC pairs (optionally, because of one or more
alterations that disfavor heterodimer formation), a MabPair mixture
comprising Ab1 and Ab2 will result.
[0459] In an alternate strategy where DNAs encoding two full-length
antibodies (Ab1 and Ab2) are introduced in the host cells, only one
of the antibodies comprises one or more HC- and/or
LC-partner-directing alteration(s), and either one or neither of
the antibodies comprises alterations that disfavor heterodimers.
See FIG. 3. Thus, in some embodiments, one of the antibodies can
comprise no partner-directing alterations and no alterations that
disfavor heterodimers, and the other can comprise one or more
partner-directing alterations and one or more alterations that
disfavor heterodimers. See FIG. 3, panel A. Alternatively, one of
the antibodies can comprise one or more partner-directing
alteration(s) and no alterations that disfavor heterodimers, while
the other antibody may comprise no partner-directing alteration(s)
but may comprise one or more alterations that disfavor
heterodimers. In a further embodiment, neither antibody comprises
an alteration that disfavors heterodimers, and only one antibody
comprises one or more partner-directing alteration. See FIG. 3,
panel B.
[0460] Further purification of an antibody mixture made by a host
cell population or host cell line can involve a number of steps. In
some embodiments, the mixture is applied to a Protein A or Protein
G affinity column and subsequently eluted. Other column
chromatography steps such as cation or anion exchange
chromatography, including low pH cation exchange chromatography as
described below, size exclusion chromatography, reverse phase
chromatography, or hydrophobic interaction chromatography (HIC)
could also be used. Further purification steps can include
diafiltration, among many possibilities.
[0461] Further, an antibody mixture or nucleic acids encoding an
antibody mixture can be formulated for its intended use. For use as
a therapeutic, the antibody mixture could be formulated as a liquid
for parenteral administration, optionally for injection. Other
kinds of formulations, e.g., gels, pastes, creams, or solids, are
also possible. Formulations can include ingredients that can, for
example, maintain, modify, or preserve the antibodies or nucleic
acids and/or control factors such as pH, osmolarity, viscosity,
clarity, odor, color, sterility, and/or rate of release or
absorption in vivo. As such, it could include any buffer and/or
excipient ordinarily used in such formulations. Examples of such
ingredients include buffers, anti-microbials, chelating agents,
salts, amino acids, and sugars, among many possibilities. The pH of
the formulated mixture could be within a range from about pH 5 to
about pH 8.5 or from about pH 6 to about pH 8.
[0462] The binding specificity of the antibodies can be determined
by a binding assay similar to that described in Example 8.
[0463] A method to determine whether two antibodies compete for a
particular binding site or epitope on an antigen generally includes
the following steps. First, a biotinylated antigen is incubated in
the presence of varying amounts of a competitor antibody (mAb2).
These combinations are referred to as "samples." The samples, which
may include mAb2/antigen complexes as well as unbound mAb2 and/or
antigen, are then added to wells in a microtiter plate coated with
another antibody that binds the antigen (mAb1). As a control,
samples including biotinylated antigen incubated without mAb2 can
be added some wells. The plate is then washed to remove unbound
antigen. If mAb1 and mAb2 do not compete, mAb1 can bind to the
mAb2/antigen complexes, as well as free antigen. In this case,
signal intensity (which is proportional to the amount of bound
antigen or mAb2/antigen complexes) will not be diminished by the
presence of mAb2 in a sample. In some cases mAb1 and mAb2 may
compete completely, meaning that mAb1 will bind to free antigen,
but not to mAb2/antigen complexes. In some cases, competition may
occur but be less complete. In such a case, binding of mAb1 to
mAb2/antigen complexes may be decreased rather than completely
absent. In either case, signal intensity will be decreased by the
presence of mAb2/antigen complexes in a sample. The signal is
detected by adding streptavidin coupled to horse radish peroxidase
(HRP), washing the plate, and adding a substrate for HRP that can
be detected by colorimetric measurements. The plate is washed, and
the reaction is stopped to prevent saturation of the signal. The
colorimetric signal is detected. As meant herein, if two antibodies
compete (either completely or partially) for binding to an antigen
by the test described here, they are said to bind to the same
epitope on the antigen.
[0464] The HCs of the antibodies in the mixture can be of any
isotype, such as IgG (including either IgG1, IgG2, IgG3, or IgG4),
IgA, IgM, IgE, or IgD. When an IgG4 HC is used, the HC can comprise
the alteration S228P, which prevents Fab arm exchange. Silva et al.
(2015), J. Biol. Chem. 290(9): 5462-5469. Sequences for such heavy
chains are known in the art. See, e.g., Kabat et al., supra, at
pages 661-723, which is incorporated herein by reference. The amino
acid sequence of the heavy chain an IgG4 antibody containing the
S228P alteration is provided in SEQ ID NO:23. The heavy chains can
be from any species, e.g., a mammal, a human, a primate, a mouse,
or a rat, or the heavy chains can be artificially produced, for
example using phage display or using a humanization process.
[0465] Similarly, the two different light chains can be lambda
(.lamda.LC) or kappa (.kappa.LC) chains, which can be from any
species and, optionally, can be mammalian, for example, human or
humanized, primate, murine, or rat antibodies. The light chain
could also be produced artificially, for example using phage
display or a humanization process. Numerous examples of amino acid
sequences of .lamda.LCs and .kappa.LCs are known in the art, for
example those reported in Kabat et al., supra, pages 647-660, which
are incorporated herein by reference. Positions in these sequences
are determined according to the Kabat (Kabat et el., supra)
numbering system for VL domains and the Edelman (Edelman et al.,
supra) numbering system for CL domains, as shown in Tables 9-11 and
discussed in the accompanying text.
[0466] Both heavy and light chains can contain one or more
alterations as described herein. Each alteration of can be a
substitution, insertion, or deletion of a single amino acid. In
some embodiments, each alteration is the substitution of one amino
acid with another. Optionally, the alteration is the substitution
of a charged amino acid or a cysteine for the amino acid originally
present at that site. In some embodiments, the substituted amino
acid can be any amino acid. In some embodiments, an amino acid
other than cysteine can be substituted for cysteine. The amino acid
other than cysteine can be any amino acid, although it can be
serine, glycine, or alanine in some embodiments. In some
embodiments the choice of the amino acid used to replace that in
the original amino acid sequence is limited. For example, in such
embodiments the amino acid used to replace the original amino acid
can be any amino acid except one or more of the following amino
acids: alanine (A), arginine (R), asparagine (N), aspartic acid
(D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G),
histidine (H), isoleucine (I), leucine (L), lysine (K), methionine
(M), phenylalanine (F), proline (P), serine (S), threonine (T),
tryptophan (W), tyrosine (Y), and valine (V). In other embodiments,
an original amino acid can be replaced with any other amino acid
from among the group of twenty recited immediately above. In other
embodiments, an original amino acid can be replaced with either of
two, three or four amino acids and/or any amino acid within a group
of amino acids having similar properties, such as the
"conservative" amino acid substitutions described below. For
example, such groups include (1) arginine and lysine, (2) serine
and threonine, (3) aspartate and glutamate, or (4) asparagine and
glutamine, among others.
[0467] One of skill in the art is aware that the amino acids
present in living things can be grouped according to their
properties and that replacement of an original amino acid with an
amino acid having similar properties is called a "conservative
substitution." The alterations described herein can, in some
embodiments, include conservative substitutions. As meant herein,
conservative substitutions include replacement of (1) A with V, L,
or I, (2) R with K, Q, or N, (3) N with Q, (4) D with E, (5) C with
S or A, (6) Q with N, (7) E with D, (8), G with P or A, (9) H with
N, Q, K, or R, (10) I with L, V, M, A, or F, (11) L with I, V, M,
A, or F, (12) K with R, Q, or N, (13) M with L, F, or I, (14) F
with L, V, I, A, or Y, (15) P with A, (16) S with T, A, or C, (17)
T with S, (18) W with Y or F, (19) Y with W, F, T, or S, and (20) V
with I, M, L, F, or A.
[0468] Amino acids and amino acid substitutions at particular sites
in a sequence are denoted herein as follows. The original amino
acid in a sequence is followed by the position number in the heavy
or light chain amino acid sequence (using the numbering systems
illustrated in Table 5-11), which is followed by the amino acid
used as a replacement. For example, K409E in an HC means that the
lysine originally present at position 409 in the HC is replaced by
glutamic acid. If position 409 in the heavy chain can originally
contain either of two different amino acids, e.g., K or R, and
these can be replaced with either of two amino acids, e.g., D or E,
that could be denoted as K/R409D/E. This designation means that the
lysine or arginine originally present at position 409 can be
replaced with either an aspartic acid or a glutamic acid. In some
cases, the original amino acid is not defined. For example, 409D in
an HC would mean amino acid 409 is an aspartic acid, and the
identity of the original amino acid is not defined and can be any
amino acid, including aspartic acid. Similarly, a designation of
K409 means that the original amino acid at position 409 is a lysine
(and there is no alteration).
[0469] Tables 5-11 illustrate the level of sequence consensus among
HCs and LCs, in some cases among human or primate HCs and LCs. The
amino acid sequences of the variable regions vary particularly in
the complementarity determining regions (CDRs, which are shown by
bold italic numbers in Tables 5 and 9). However, the framework
regions that surround the CDRs are more conserved and contain
highly conserved amino acids at a number of positions. Many of the
universally conserved or almost universally conserved amino acids,
for example positions 4, 36, 38, and 39 in VH and 98, 99, 101, 102
in VL, are also conserved in VH and VL regions from non-human
species. In addition, many other sites in VHs and VLs are highly
conserved within specific groups of variable domains, although not
across all VHs and VLs. Constant domains in HCs and LCs show a
higher degree of sequence conservation than variable domains and
contain a number of highly conserved amino acids. See Tables 6-8
and 10-11. Using these highly conserved amino acids, one of skill
in the art would be able to align most immunoglobulin domains with
the sequences disclosed in Kabat et al., supra to assign a
numbering of those VHs and VLs according to the system of Kabat et
al., supra or Edelman et al., supra.
[0470] The antibodies in the mixtures described herein can comprise
HC- and/or LC-partner-directing alterations. HC- and/or
LC-partner-directing alterations serve the function of ensuring
that each LC pairs with its cognate HC and vice versa. In the
absence of such alterations, up to ten different species of
antibodies could potentially form in a host cell transfected with
DNA encoding only two different full-length antibodies that have
different HCs and LCs. See FIG. 4. Of course, many more species
could potentially form in a host cell transfected with DNA encoding
three or four different full-length antibodies having different HCs
and LCs and lacking partner-directing alterations. However, if only
cognate HC/LC pairing occurs, these numbers would be drastically
reduced. If only homodimeric HC/HC pairing occurs, the number of
species would be further reduced. Since many of the possible
species in the absence of partner-directing alterations would have
non-cognate HC/LC pairings, some of the resulting antibodies might
not bind to any epitope and might therefore lack a desired
function. Given the very strict requirements for uniformity of a
pharmaceutical product, such a complex mixture is unlikely to
receive regulatory approval as a therapeutic.
[0471] HC- and LC-partner-directing alterations can occur at
contacting sites in the CH1 and CL domains, which are listed in
Table 12 above, and/or at contacting sites in VH and VL domains, as
explained herein. Antibodies in the mixtures described herein can
comprise one or more LC- and/or HC-partner-directing alteration(s)
in their HC and/or LC, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 such alterations and/or
not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 such alterations. In some embodiments, at
least one antibody in the mixture can lack such alterations.
[0472] In some embodiments, these alterations include a
substitution of a charged amino acid at a site that did not
originally have a charged amino acid or substitution of a charged
amino acid at a site that originally contained an amino acid of
opposite charge. In embodiments where DNA encoding two different
full-length antibodies, where the HCs and the LCs of the two
antibodies are different, is introduced into a host cell, the LC-
and HC-partner-directing alterations can occur at the same sites in
the two different LCs and HCs, resulting in a situation where (1)
the two different LCs have oppositely charged amino acids at the
same LC site, (2) the two different HCs have oppositely charged
amino acids at an HC site contacting the LC site, and (3) for each
cognate LC/HC pairing, the charged amino acid at the LC site is
opposite in charge to the amino acid at the contacting HC site. In
such a situation, the interaction between cognate LCs and HCs is
strengthened by an interaction between oppositely-charged amino
acids, and the interaction between non-cognate LC/HC pairings is
highly disfavored due to repulsion of amino acids having the same
charges situated at contacting sites in non-cognate HC/LC pairs.
See FIGS. 1-2. In other embodiments, only one antibody comprises a
charge pair that favors cognate HC/LC pairing. See FIG. 3. In such
a case, one of the amino acids in the charge pair may result from a
partner-directing alteration where a charged amino acid residue is
substituted for an oppositely charged amino acid residue. In this
case, some non-cognate HC/LC pairs would be expected to be
disfavored due to repulsive charge interactions. Examples of
contacting charge pairs optionally resulting from HC- and/or
LC-partner-directing alterations include, without limitation, the
following: 44D/E (HC) and 100R/K (LC); 44R/K (HC) and 100D/E (LC);
105E/D (HC) and 43R/K (LC); 105R/K (HC) and 43E/D (LC); 133R/K (HC)
and 117D/E (LC); 133D/E (HC) and 117R/K (LC); 137R/K (HC) and
114D/E (LC); 137D/E (HC) and 114R/K (LC); 137R/K (HC) and 116D/E
(LC); 137D/E (HC) and 116R/K (LC); 147R/K (HC) and 124D/E (LC);
147D/E (HC) and 124R/K (LC); 147R/K (HC) and 129D/E (LC); 147D/E
(HC) and 129R/K (LC); 147R/K (HC) and 131D/E (LC); 147D/E (HC) and
131R/K (LC); 147R/K (HC) and 178D/E (LC); 147D/E (HC) and 178R/K
(LC); 147R/K (HC) and 180D/E (LC); 147D/E (HC) and 180R/K (LC);
168D/E (HC) and 164R/K (LC); 168R/K (HC) and 164D/E (LC); 168D/E
(HC) and 167R/K (LC); 168R/K (HC) and 167D/E (LC); 168D/E (HC) and
174R/K (LC); 168R/K (HC) and 174D/E (LC); 170D/E (HC) and 162R/K
(LC); 170R/K (HC) and 162D/E (LC); 173D/E (HC) and 160R/K (LC);
173R/K (HC) and 160D/E (LC); 173D/E (HC) and 162R/K (LC); 173R/K
(HC) and 162D/E (LC); 175D/E (HC) and 160R/K (LC); 175R/K(HC) and
160D/E (LC); 175D/E (HC) and 180R/K (LC); 175R/K (HC) and 180D/E
(LC); 176D/E (HC) and 160R/K (LC); 176R/K (HC) and 160D/E (LC);
181R/K (HC) and 178D/E (LC); 181D/E (HC) and 178R/K (LC); 183R/K
(HC) and 176D/E (LC); 183D/E (HC) and 176R/K (LC); 190R/K (HC) and
116D/E (LC); 190D/E (HC) and 116R/K (LC); 190R/K (HC) and 137D/E
(LC); 190D/E (HC) and 137R/K (LC).
[0473] In, for example, embodiments where DNA encoding two
full-length antibodies (a first antibody comprising HC1 and LC1 and
a second antibody comprising HC2 and LC2) has been introduced into
a host cell, one or both sites in a pair of contacting sites in an
HC/LC pair may already contain a charged amino acid. For example,
in some cases LC1 may comprise a charged amino acid at a site that
contacts a site in the cognate HC1 that does not comprise a charged
amino acid, or vice versa. In a specific example, position 160 in a
human CL domain can comprise a glutamic acid (E), whereas
contacting sites 173, 174, and 175 in a human CH1 domain commonly
comprise V, L, and Q, respectively. In this case, one of the
contacting CH1 sites, e.g., 173, can be altered such that it is
opposite in charge to that of the E at position 160 in the CL
domain, i.e. position 173 can be substituted with an R or a K. The
same site in HC2 can be altered to a charged amino acid opposite in
charge to that at the site in HC1 (i.e., this site can be
substituted with a D or an E), and the contacting site in LC2 can
be altered to a charged amino acid opposite in charge to that in
LC1 (i.e., position 160 in LC2 can be substituted with an R or a
K).
[0474] Other scenarios where one or more of the contacting sites in
a cognate HC/LC pair already comprise one or more charged amino
acid(s) could be treated similarly with the end goal of creating
oppositely charged amino acids at contacting sites in cognate HC/LC
pairs. In some embodiments, the same pair of contacting sites can
be altered such that they comprise oppositely charged amino acids
in both antibodies, with the proviso that the charges of the amino
acids at the HC site is opposite in HC1 and HC2 and the charges at
the LC site are also opposite in the LC1 and LC2.
[0475] Similarly, for example in embodiments where DNA encoding two
full-length antibodies (a first antibody comprising HC1 and LC1 and
a second antibody comprising HC2 and LC2) has been introduced into
a host cell, if contacting sites in HC1 and LC1 comprise oppositely
charged amino acids, the same sites in HC2 and LC2 can be replaced
with amino acids opposite in charge to those found in HC1 and LC1,
respectively.
[0476] In other embodiments where DNA encoding two different
full-length antibodies is introduced into a host cell, the LC- and
HC-partner-directing alterations can occur at different sites in
the two different LCs and HCs, resulting in a situation where (1)
one LC and its cognate HC each have oppositely-charged amino acids
at contacting sites and (2) the other LC and its cognate HC each
have oppositely-charged amino acids at contacting sites that differ
from those used in the first LC/HC pair. In these embodiments, the
HC- and LC-partner-directing alterations serve to strengthen the
interaction between cognate HC/LC pairs.
[0477] In still other embodiments, one or more of the antibodies in
a mixture as described herein may not comprise a partner-directing
alteration. In such embodiments, another of the antibodies in the
mixture can comprise one or more partner-directing alterations.
[0478] In other embodiments, HC- and LC-partner-directing
alterations can result in the creation of disulfide bridges due to
cysteine substitutions at contacting sites in cognate HC/LC pairs.
In, for example, embodiments where DNA encoding two different
full-length antibodies is introduced into a host cell, such LC- and
HC-partner-directing alterations can occur at different sites in
the two different LCs and HCs, resulting in a situation where
cognate LC/HC pairs have cysteine substitutions at contacting
sites, whereas non-cognate LC/HC pairs do not have cysteine
substitutions at contacting sites. See FIGS. 1-3. Thus, the two
different cognate HC/LC pairs will have disulfide bridges at
different places at the HC/LC interface, whereas a non-cognate
HC/LC pair would not have such a disulfide bridge because the
substituted cysteine residues would not be close enough to form a
bridge. Hence, in these embodiments, the HC- and
LC-partner-directing alterations serve to strengthen the
interaction between cognate HC/LC pairs. In other embodiments, one
or more antibodies in a mixture, but not all antibodies in the
mixture, can comprise cysteines at contacting sites in a cognate
HC/LC pair. Examples of pairs of cysteine substitutions at
contacting residues include, for example, the following pairs of
alterations: 126C (HC) and 121C (LC); 126C (HC) and 124C (LC); 127C
(HC) and 121C (LC); 128C (HC) and 118C (LC); 133C (HC) and 117C
(LC); 133C (HC) and 209C (LC); 134C (HC) and 116C (LC); 141C (HC)
and 116C (LC); 168C (HC) and 174C (LC); 170C (HC) and 162C (LC);
170C (HC) and 176C (LC); 173C (HC) and 160C (LC); 173C (HC) and
162C (LC); and 183C (HC) and 176C (LC).
[0479] In some embodiments, one or more cysteine residues that
normally form part of a disulfide bridge between an HC and an LC
can be replaced with another amino acid in at least one of the
antibodies in a mixture as described herein. For example, in a
human IgG1 antibody, the cysteines at position 220 in the HC and
214 in the LC form a disulfide bridge between the HC and LC. These
amino acids can be replaced with other amino acids, for example
serine, alanine, or glycine, thereby eliminating an HC/LC disulfide
bridge. Similar alterations can be made in antibodies of other IgG
isotypes, i.e., IgG2, IgG3, or IgG4, with similar or different
patterns of disulfide bond formation, in which the cysteine
residues that participate in HC/LC disulfide bond formation can be
substituted with other amino acids. For example, in human IgG2 and
IgG4 antibodies, the cysteines at positions 131 (HC) and 214 (LC)
can be substituted with other amino acids. Such alterations can
weaken non-cognate HC/LC pairing, as well as cognate HC/LC pairing,
since non-cognate pairs will also be unable to form the usual
interchain disulfide bridges. Cognate HC/LC pairing can be
strengthened by, e.g., adding partner-directing alterations to the
cognate HC/LC pair lacking it usual disulfide bridge(s). Such
partner-directing alterations can include cysteine substitutions at
contacting residues in the HC and/or the LC so as to create new
disulfide bridges and/or substitutions that introduce charged amino
acids at contacting residues in the HC and/or LC so as to create
charge pairs. See FIG. 3.
[0480] Where DNA encoding at least two and not more than four
different full-length antibodies having different HCs and LCs has
been introduced into a host cell, if the HCs of the antibodies do
not contain any alterations to disfavor the formation of
heterodimers, heterodimeric HC/HC pairings can generally occur. If
DNA encoding only two different full-length antibodies has been
introduced and only cognate HC/LC pairs can form (due to HC- and/or
LC-partner directing alterations), this will lead to the formation
of three different major antibody species (called "3-in-1 mixture"
herein), the two starting antibodies plus a bispecific antibody
comprising a cognate HC/LC pair from each of the starting
antibodies. See FIGS. 2-4. As explained below with reference to a
mixture containing two anti-HER2 antibodies that bind to different
epitopes, this may be an advantageous situation for some
mixtures.
[0481] In some embodiments where DNA encoding two different
full-length antibodies having different HCs and LCs has been
introduced into a host cell, the HCs of at least one of the two
antibodies can comprise one or more alteration(s) that disfavors
the formation of HC/HC heterodimeric pairings. If essentially only
homodimeric HC/HC pairs can form and essentially only cognate HC/LC
pairs can form (due to HC- and/or LC-partner-directing alterations
and/or the elimination of HC/LC disulfide bridges in at least one
of the antibodies), this will lead to the formation of only two
different major antibody species (called "MabPair" mixtures
herein), the two starting full-length antibodies. See FIGS. 1 and
3. In the context of a pharmaceutical production process, this
leads to the advantageous situation where a pharmaceutical product
consisting essentially of two different antibodies can be produced
in a single cell line using a single production process. Examples
of alterations (including in some cases amino acids present in the
original sequence) that disfavor the formation of heterodimers
include, without limitation, the following: 409D/E plus 399R/K in
one HC where the other HC has an arginine at position 409 (e.g., as
in a human IgG4 antibody); 409D/E, 399R/K, plus 392D/L/Y/M/W/I/V/F
in one HC where the other HC has an arginine at position 409;
392E/D plus 399R/K in one HC and 392K/R plus 399D/E other HC;
399K/R, 409D/E, plus 392D/E in one HC and 399D/E, 409K/R, plus
392K/R in the other HC; 399K/R, 409D/E, 356K/R, plus 392D/E in one
HC and 399D/E, 409K/R, 356D/E, plus 392K/R in the other HC; 399K/R,
409D/E, 357K/R, plus 392D/E in one HC and 399D/E, 409K/R, 357D/E,
plus 392K/R in the other HC; 399K/R, 409D/E, 356K/R, plus 438D/E in
one HC and 399D/E, 409K/R, 356D/E, plus 438K/R in the other HC;
399K/R, 409D/E, 357K/R, plus 370D/E in one HC and 399D/E, 409K/R,
357D/E, plus 370K/R in the other HC; 399K/R, 409D/E, 356K/R,
392D/E, 357K/R, plus 370D/E in one HC and 399D/E, 409K/R, 356D/E,
392K/R, 357D/E, plus 370K/R in the other HC; 399K/R, 409D/E,
356K/R, 392D/E, 357K/R, plus 439D/E in one HC and 399D/E, 409K/R,
356D/E, 392K/R, 357D/E, plus 439K/R in the other HC; 366Y/W plus
407T/A in one HC and 366T/A plus 407Y/W in the other HC; 405A/T
plus 394W/Y in one HC and 405W/Y plus 394A/T in the other HC;
407Y/W in one HC and 366Y/W in the other HC; 366Y/W, 407T/A, plus
405A/T, 394W/Y in one HC and 366T/A, 407Y/W, plus 405Y/W, plus
394A/T in the other; and 366W/Y, 368A/T, plus 407V/T/A in one HC
and 366T/A/S, 368L, plus 407Y/W in the other HC.
[0482] In further embodiments, one or more alterations that affect
the pharmacokinetic properties of one or more of the antibodies a
mixture as described herein can be introduced. For example, the in
vivo half life or the area under the curve (AUC) can be shortened
by alterations such as M252A, M252L, M252S, M252R, R255K or H435R.
Other alterations that affect pharmacokinetic properties of one or
more antibody in an antibody mixture can be introduced.
Antibodies
[0483] Described herein are antibodies that comprise
partner-directing alterations described herein. Such antibodies can
be antibodies of any format that comprises VH, CH1, VL, and CL
domains. In some embodiments, such antibodies can be full-length
IgG antibodies that can be IgG1, IgG2, IgG3, or IgG4 antibodies,
which can be mammalian antibodies, e.g., primate, human, and/or
humanized antibodies. These include, for example, an antibody
comprising, for example, a primate, human, and/or humanized CL and
IgG1 CH1 domains that comprise one or more charge pairs (which can
result from partner-directing alteration(s)) at one or more of the
following pairs of sites in the HC and LC, respectively: 147 and
131; 168 and 174; and 181 and 178. Further embodiments include an
antibody comprising, for example, primate, human, and/or humanized
CL and IgG4 CH1 domains that comprise one or more charge pairs
(which can result from partner-directing alteration(s)) at one or
more of the following pairs of sites in the HC and LC,
respectively: 147 and 131; 168 and 174; and 181 and 180. In further
embodiments, described herein are an antibody comprising, for
example, primate, human, and/or humanized CL and IgG1 CH1 domains,
wherein the antibody comprises one or more pairs of cysteine
residues at contacting sites in the CH1 and CL domains, wherein the
CH1 and CL positions, respectively, of these cysteine residues can
be at any one or more of the following pairs: 126 and 124; 128 and
118; 133 and 117; 134 and 116; 168 and 174; 170 and 162; 170 and
176; and 173 and 160. In still other embodiments, described herein
are an antibody comprising, for example, primate, human, and/or
humanized CL and IgG4 CH1 domains, wherein the antibody comprises
one or more pairs of cysteine residues at contacting sites in the
CH1 and CL domains, wherein the CH1 and CL positions, respectively,
of these cysteine residues can be at any one or more of the
following pairs of residues: 126 and 124; 127 and 121; 128 and 118;
168 and 174; 170 and 162; and 173 and 162;
[0484] In a further embodiment, described herein is an IgG2
antibody, optionally a human, primate, and/or humanized antibody,
lacking the naturally occurring disulfide bridge linking the HC and
LC and containing one or more substitutions in both the HC and the
LC that can create one or more new disulfide bridge. For example,
the cysteine at position 131 in a human, humanized, and/or primate
IgG2 HC can be replaced with another amino acid, e.g., serine,
alanine, or glycine, and the cysteine at position 214 in the
cognate LC can be replaced with another amino acid, e.g., serine,
alanine, or glycine. These substitutions would eliminate the
naturally occurring disulfide bridge between an IgG2 HC and its
cognate LC. A new disulfide bridge could be created by introducing
a cysteine substitution at each residue of a pair of contacting
residues, where one residue is in the IgG2 HC and other is in the
LC. For example, the substitutions F170C in the HC and S162C in the
LC are such a pair of cysteine substitutions at contacting
residues, as are V173C in the HC and 0160C in the LC. Other
cysteine substitutions at other pairs of contacting residues could
also be used. This approach could avoid the formation of multiple
IgG2 structural isomers due to disulfide bond shuffling, which has
been observed in native human IgG2 antibodies. See, e.g., Lightle
et al. (2010), Protein Science 19: 753-762. Formation of multiple
structural isomers can be disadvantageous when manufacturing an
antibody for use as a therapeutic since a homogeneous preparation
is generally preferred.
[0485] Any of the antibodies described above can be made using
standard methods in the art. For example, an antibody can be made
by (1) introducing one or more DNAs encoding the antibody,
optionally in one or more appropriate vectors, into host cells, (2)
culturing the host cells under conditions conducive to expression
of the antibody, and (3) obtaining the antibody from the cell
supernatant or host cell mass.
Target Molecules Bound by the Antibodies and/or Mixtures of
Antibodies
[0486] The different antibodies in the mixtures described herein
bind to different epitopes and can bind to one or more target
molecule. The target molecules, optionally proteins, for antibodies
and/or antibody mixtures described herein can be chosen in light of
knowledge of the role of various molecules in a disease state. In
some embodiments, the disease is a human disease, and the target
molecule(s) is (are) one or more human protein(s). Similarly, the
antibodies described above can bind to one of these target
molecules.
[0487] In one example, the target protein(s) for an antibody
mixture can be one or more protein(s) that serve(s) as a checkpoint
that inhibits or blocks the activity of the immune system. Since
cancers and infections can be recognized by the immune system and
the immune system may regulate and even eliminate tumors and
infections, preventing regulation or blockage of immune system
activity could potentially limit growth of cancer cells or
eliminate infections, in some embodiments, viral infections.
Checkpoint-blocking antibodies, such as those directed against
cytotoxic T-lymphocyte antigen 4 (CTLA4) and programmed death 1
receptor (PD1), have demonstrated promise in the treatment of an
expanding list of malignancies. While both CTLA4 and PD1 function
as negative regulators, each plays a non-redundant role in
modulating immune responses. CTLA4 attenuates the early activation
of naive and memory T cells, and PD1 is primarily involved in
modulating T cell activity in peripheral tissues via interaction
with its ligands, PD-L1 and PD-L2. A single antibody could also
bind to any of these target proteins.
[0488] Accumulating clinical evidence points toward a promising
role for checkpoint-blocking antibodies in a rapidly expanding
spectrum of solid tumors, including non-small cell lung cancer,
renal cell cancer, ovarian cancer, bladder cancer, head and neck
cancer, and gastric cancer. While blocking either the CTLA4 or the
PD1 pathway inhibits growth of multiple tumor types, the overall
response rate is still low, underscoring the importance of
improving upon present options. Combined checkpoint blockade, to
date explored with anti-CTLA4 (ipilimumab) and anti-PD1 (nivolumab)
pathway blocking agents, has shown better clinical efficacy than
ipilimumab alone or nivolumab alone in patients with untreated
melanoma. Larkin et al. (2015), New Engl. J. Med. 373: 23-34. In
melanoma patients with programmed cell death 1 ligand (PD-L1; also
known as PDCD1LG1, PDCD1L1, B7H1, and CD274)-positive tumors,
progression-free survival using treatment with nivolumab alone was
essentially the same as that observed using treatment with
nivolumab plus ipilimumab and was higher than that observed using
treatment with ipilimumab alone. Larkin et al., supra. In patients
with PD-L1-negative tumors, combination therapy resulted in longer
progression-free survival than was observed with nivolumab or
ipilimumab alone. Larkin et al, supra. These data provide a strong
rationale for anti-cancer therapeutics that include two or more
antibodies that block two or more immune system checkpoint
proteins. Similar studies using another anti-PD1 antibody,
prembrolizumab, are currently ongoing. Such mixtures could be made
using the methods described herein to make, for example, a MabPair
mixture or a 3-in-1 antibody mixture.
[0489] In the context of making mixtures of different immune
checkpoint-blocking antibodies, the isotype of the antibodies can
be of importance because different isotypes can elicit different
effector functions. Of the five immunoglobulin isotypes,
immunoglobulin G (IgG) is most abundant in human serum. The four
IgG subclasses, IgG1, IgG2, IgG3, and IgG4, differ in their
constant regions, especially in their hinge and upper CH2 domains.
These regions are involved in binding to IgG-Fc receptors
(Fc.gamma.R), which can initiate antibody-dependent cell-mediated
cytotoxicity (ADCC) and/or phagocytosis (ADCP), and Clq, which can
initiate complement dependent cytotoxicity (CDC). Hence, the
different subclasses have different effector functions. IgG1 and
IgG3 antibodies can elicit potent effector responses including
ADCC, ADCP, and CDC, whereas IgG2 and IgG4 antibodies elicit much
more subtle effector responses and only do so in certain cases.
Antibody responses to soluble protein antigens and membrane
proteins primarily induce production of IgG1 antibodies,
accompanied by lower levels of other IgG subclasses, mostly IgG3
and IgG4. Ferrante et al. (1990), Pediatr. Infect. Dis. J. 9(8
Suppl):516-24.
[0490] For therapeutic antibodies, IgG1 has been the most popular
choice by far. Antibodies designed for selective eradication of
cancer cells typically require an isotype that can elicit potent
complement activation and effector-mediated cell killing by ADCC.
Although IgG1 and IgG3 both meet these criteria, IgG3 has not been
used for therapeutic antibody development, probably because of a
shorter half-life, susceptibility of the relatively long hinge
region to proteolysis, and extensive allotypic polymorphism.
[0491] Antibody isotype can be an important consideration for
anti-CTLA4 antibodies used to treat cancer. Preclinical data
suggests that a checkpoint-blocking anti-CTLA4 antibody might
deliver much of its therapeutic effect through killing of T
regulatory (Treg) cells within tumors, thus releasing CD8 T
cell-mediated anti-tumor immunity. Simpson et al. (2013), J Exp
Med. 210(9): 1695-1710. Similar mechanisms may operate in human
patients. Ipilimumab, a human IgG1 anti-CTLA4 antibody, was
recently shown to lead to ADCC-mediated lysis of human Tregs ex
vivo. Romano et al. (2015), Proc. Natl. Acad. Sci. 112(19):
6140-6145. Further, in a small clinical study, melanoma patients
responding to ipilimumab had significantly higher baseline
frequencies of nonclassical monocytes and more activated
tumor-associated macrophages expressing Fc.gamma.RIII, which
correlated with lower intratumoral Treg numbers after therapy,
suggesting that Treg deletion occurs in these patients. Therefore,
an IgG1 or IgG3 isotype may be favored for an anti-CTLA4 antibody
used in the treatment of cancer since the antibody might have the
greatest effect if it causes potent killing of the Treg cells
within the tumor.
[0492] The IgG isotype of choice for anti-PD1 antibodies is
typically IgG4 or a mutated IgG1 with minimal Fc.gamma.R
interactions. PD1 is expressed on the surface of activated T cells,
B cells, and macrophages, and negatively regulates immune
responses. Since PD1 is expressed on these effector cells, it may
not be desirable to use an isotype that can elicit strong effector
functions, i.e., IgG1 or IgG3, because this could result in killing
activated T cells, which might otherwise kill cancer cells.
[0493] Hence, in making an anti-cancer therapeutic containing a
mixture of an anti-CTLA4 and an anti-PD1 antibody, the inclusion of
a bispecific anti-CTLA4 anti-PD1 antibody may be inappropriate
because, as explained above, different effector functions are
appropriate for each of the two binding domains. Moreover, the
effects of bringing regulatory T cells and effector T cells into
close physical proximity by means of an anti-PD1 and anti-CTLA4
bispecific antibody are unpredictable. A mixture consisting
essentially of a monospecific IgG1 anti-CTLA4 antibody and a
monospecific IgG4 (or modified IgG1) anti-PD1 antibody may be
desirable. Such a MabPair mixture could be made in a single host
cell as described herein using alterations to disfavor heterodimers
and LC- and HC-partner-directing alterations described herein.
[0494] Moreover, other combinations of immune checkpoint proteins
could serve as targets for MabPair mixtures or 3-in-1 mixtures of
antibodies. In situations where different isotypes are appropriate
for each binding domain (as in the case of CTLA4 and PD1), a
MabPair mixture could be preferred so that each binding domain
would be attached to the desired constant region in most or all
cases. In cases where the same isotype is appropriate for both
binding domains, either a MabPair mixture or a 3-in-1 mixture could
be used.
[0495] In another example, the human epidermal growth factor
receptor (HER) family of proteins, i.e., HER1, HER2, HER3, and
HER4, plays an important role in cell survival and proliferation
and has been implicated in oncogenesis. These proteins are capable
of forming heterodimers and homodimers, which can activate signal
transduction pathways that regulate many cellular processes,
including growth, proliferation, and survival. Overexpression of
HER2 is associated with aggressive disease and poor prognosis in
human breast cancer patients. Treatment of such patients with
anti-HER2 antibodies, such as HERCEPTIN.RTM. (the brand name for a
humanized anti-HER2 antibody called trastuzumab), with or without
lapatinib (a small molecule tyrosine kinase inhibitor), has
improved survival. Trastuzumab binds domain IV of HER2 and inhibits
HER2-mediated cell proliferation by activating antibody-dependent
cellular cytotoxicity (ADCC), preventing formation of p95HER2 (a
truncated and constitutively active form of HER2), blocking
ligand-independent HER2 signaling, and inhibiting HER2-mediated
angiogenesis.
[0496] A different humanized anti-HER2 antibody called pertuzumab
binds to a different epitope (in domain II of HER2) than
trastuzumab and inhibits HER2 dimerization with other HER family
members such as HER3 and HER1, thus inhibiting the downstream
signaling processes that are associated with tumor growth and
progression. The combination of pertuzumab and trastuzumab has a
strongly enhanced antitumor effect compared to either agent alone
and induces tumor regression in xenograft models (Yamashita-Kashima
(2011), Clin. Cancer Res. 17(15): 5060-5070; Scheuer et al. (2009),
Cancer Res 69: 9330-9336), something that cannot be achieved by
either monotherapy. The enhanced efficacy of the combination was
also observed after tumor progression during anti-HER2 trastuzmab
monotherapy. Binding of pertuzumab to tumors is not impaired by
trastuzumab pretreatment. Furthermore, both trastumab and
pertuzumab potently activate ADCC. The strongly enhanced antitumor
activity is likely due to the differing and complementary
mechanisms of action of trastuzumab and pertuzumab. Potentially, a
bispecific antibody that could bind to both epitopes on one or more
molecules of HER2 protein simultaneously might have different
activity, possibly greater or lesser, than the two separate
antibodies. In some cases, a bispecific antibody binding to two
different epitopes on a single target protein might not be able to
simultaneously bind to the two epitopes on a single target protein.
Thus, there is reason to believe that treatment with two or more
anti-HER2 antibodies that bind to different epitopes, optionally
including a bispecific antibody, can be more effective than
treatment with a single antibody.
[0497] Hence, the methods described herein could be used to make
mixtures containing two or more different anti-HER2 antibodies. For
example, a MabPair mixture or a 3-in-1 antibody mixture could be
made. Such mixtures could be used to treat a subset of breast
cancer patients, i.e., those with cancers that overexpress HER2,
and possibly other cancer patients with HER2-mediated cancers.
[0498] Further, the methods described herein could be used to make
mixtures of antibodies that bind to other cancer antigens, i.e.,
proteins that are overexpressed on cancer cells. In most of these
cases, IgG1 and/or IgG3 isotype(s) would be desirable because
killing of the cancer cells is a therapeutic objective. For
example, the antibodies in the mixtures could bind to different
epitopes on a single cancer antigen. Alternatively, the antibodies
in the mixtures could bind to different cancer antigens if the
cancer cells express multiple different cancer antigens. In either
case, the mixtures could be, for example, MabPair or 3-in-1
mixtures, depending on the biological role of the target
proteins.
[0499] Examples of pairs of target molecules for the antibody
mixtures described herein include, without limitation, the
following pairs of target proteins (shown as first target
protein/second target protein), which can be human proteins:
PD1/CTLA4, PD1/lymphocyte activation gene 3 (LAGS),
PD1/glucocorticoid-induced tumor necrosis factor receptor-related
gene (GITR; also known as AITR or TNFRSF18), PD1/vascular
endothelial growth factor A (VEGF; also known as VEGFA),
PD1/colony-stimulating factor 1 receptor (CSF1R; also known as FMS,
c-FMS and CD115), PD1/OX40 (also known as TNFRSF4, ACT35, and
CD134), PD1/T-cell immunoreceptor with immunoglobulin and ITIM
domains (TIGIT), PDL1/CTLA4, PDL1/VEGF, PDL1/OX40, PDL1/CSF1R,
PDL1/TIGIT, PDL1/T-cell immunoglobulin and mucin domains-containing
protein 3 (TIM3, also known as HAVCR2), CTLA4/VEGF, CTLA4/41BB
(also known as TNFRSF9, ILA, and CD137), membrane-spanning 4
domains, subfamily A, member 1 (CD20; also known as MS4A1 and
B1)/leukocyte surface antigen CD37 (CD37), angiopoietin 2 (ANG2;
also known as ANGPT2)/VEGF, tumor necrosis factor (TNF; also known
as TNFA and cachetin)/interleukin 17a (IL17a; also known as CTLA8),
CD38 antigen (CD38)/CD138 antigen (CD138; also known as SDC1 and
SYND1), epidermal growth factor receptor (EGFR; also known as
ERBB1, HER1, and SA7)/HER2, EGFR/HER3, MET protooncogene (MET; also
known as HGFR)/VEGF, MET/EGFR, thymic stromal lymphopoietin
(TSLP)/interleukin 33 (IL33; also known as C90RF26, NFHEV, and
IL1F11), interleukin 4 (IL4; also known as BSF1)/interleukin 13
(IL13), HER2/HER2, PD1/CD96, PD1/Protein-tyrosine phosphatase,
nonreceptor type, substrate-1 (also known as SIRP-alpha-1, PTPNS1,
SIRPA, SHPS1, MYD1, and MFR), and PD1/Chemokine, CC motif, receptor
8 (also known as CCR8, Chemokine, CC motif, receptor-like 2
(CMKBRL2), Chemokine receptor-like 1 (CKRL1), and CMKBR8). Single
antibodies could also bind to any of these target molecules.
[0500] The examples of particular targets discussed above are
exemplary. Antibodies and/or antibody mixtures that bind to any
target or combination of these targets could be made using methods
described herein.
Nucleic Acids and Vectors Encoding Antibodies and Mixtures of
Antibodies
[0501] Provided are nucleic acids, e.g., DNA, encoding the
antibodies and mixtures of antibodies described herein. Numerous
nucleic acid sequences encoding immunoglobulin domains, for example
VH, VL, hinge, CH1, CH2, and CH3 domains are known in the art. See,
e.g., Kabat et al., supra. Using the guidance provided herein, one
of skill in the art could combine known or novel nucleic acid
sequences encoding antibodies and modify them by known methods to
create nucleic acids encoding the antibodies and mixtures of
antibodies described herein, which comprise alterations as
described herein.
[0502] Methods of modifying nucleic acids are well-known in the
art. Perhaps the most straightforward method for creating a
modified nucleic acid is to synthesize a nucleic acid having the
desired sequence. A number of companies, e.g., DNA 2.0 (Menlo Park,
Calif., USA), BlueHeron (Bothell, Wash.), Genewiz (South
Plainfield, N.J.), Gen9 (Cambridge, Mass.), and Integrated DNA
Technologies (Coralville, Iowa; IDT), provide this service. Other
known methods of introducing mutations, for example site-directed
mutagenesis using polymerase chain reaction (PCR), can also be
employed. See, e.g., Zoller (1991), Curr. Opin. Biotechnol. 2(4):
526-531; Reikofski and Tao (1992), Biotechnol. Adv. 10(4): 535-547.
For example, Example 2 below describes the use of a commercial kit
to introduce specific mutations into a starting DNA.
[0503] The DNA vector(s) that contain(s) the DNA encoding the HCs
and LCs of the antibodies can be any vector(s) suitable for
expression of the antibodies in a chosen host cell. The vector can
include a selectable marker for selection of host cell cells
containing the vector and/or for maintenance and/or amplification
of the vector in the host cell. Such markers include, for example,
(1) genes that confer resistance to antibiotics or other toxins,
e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host
cells, (2) genes that complement auxotrophic deficiencies of the
cell, or (3) genes whose operation supplies critical nutrients not
available from complex or defined media. Specific selectable
markers include the kanamycin resistance gene, the ampicillin
resistance gene, and the tetracycline resistance gene. A neomycin
resistance gene may also be used for selection in both prokaryotic
and eukaryotic host cells. A dihydrofolate reductase (DHFR) gene
and/or a promoterless thymidine kinase gene can be used in
mammalian cells, as is known in the art.
[0504] In addition, a vector can contain various other sequence
elements necessary for the maintenance of the vector and/or the
expression of the inserted sequences encoding the antibodies and/or
antibody mixtures described herein, e.g., the HCs and LCs of the
antibody mixtures described herein. Such elements include, for
example, an origin of replication, a promoter, one or more
enhancers, a transcriptional terminator, a ribosome binding site, a
polyadenylation site, and a polylinker insertion site for exogenous
sequences (such as the DNA encoding the antibody mixtures described
herein). These sequence elements can be chosen to function in the
desired host cells so as to promote replication and/or
amplification of the vector and expression and of the heterologous
sequences inserted into the vector. Such sequence elements are well
known in the art and available in a large array of commercially
available vectors. Many vectors are commercially available from
companies including Promega Corporation (Madison, Wis., USA) and
Agilent Technologies (Santa Clara, Calif., USA), among many
others.
[0505] DNA encoding each of two or more antibodies can be
introduced into a population of host cells using any appropriate
method including, for example, transfection, transduction,
transformation, bombardment with microprojectiles, microinjection,
or electroporation. In some embodiments, DNA encoding two
full-length IgG antibodies is introduced into the host cells. Such
methods are known in the art and described in, e.g., Kaestner et
al. (2015), Bioorg. Med. Chem. Lett. 25: 1171-1176, which is
incorporated herein by reference.
[0506] In some embodiments, nucleic acids encoding an antibody or a
mixture of antibodies can be carried on one or more viral vectors.
Examples of such viral vectors include adenovirus, adeno-associated
virus (AAV), retrovirus, vaccinia virus, modified vaccinia virus
Ankara (MVA), herpes virus, lentivirus, or poxvirus vectors. In
such embodiments, these viral vectors containing nucleic acids
encoding the mixtures or antibodies can be administered to patients
to treat a disease. In a cancer patient, such viral vectors
containing nucleic acids encoding the mixture of antibodies can be
administered directly to a tumor or a major site of cancer cells in
the patient, for example by injection, inhalation (for a lung
cancer), topical administration (for a skin cancer), and/or
administration to a mucus membrane (through which the nucleic acids
can be absorbed), among many possibilities.
[0507] Similarly, nucleic acids encoding a mixture of antibodies as
described herein, which can be encased in liposomes, can be
administered to a patient suffering from a disease.
Host Cells that Can Produce Mixtures of Antibodies
[0508] The host cells into which DNA(s) encoding antibodies are
introduced can be any of a variety of cells suitable for the
expression of a recombinant protein. These include, for example,
gram negative or gram positive prokaryotes, for example, bacteria
such as Escherichia coli, Bacillus subtilis, or Salmonella
typhimurium. In other embodiments, the host cells can be eukaryotic
cells, including such species as Saccharomyces cerevisiae,
Schizosaccharomyces pombe, or eukaryotes of the genus
Kluyveromyces, Candida, Spodotera, or any cell capable of
expressing heterologous polypeptides. In further embodiments, the
host cells can be mammalian cells. Many mammalian cells suitable
for expression of heterologous polypeptides are known in the art
and can be obtained from a variety of vendors including, e.g.,
American Type Culture Collection (ATCC). Suitable mammalian cells
include, for example, the COS-7 line (ATCC CRL 1651) (Gluzman et
al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL
163), Chinese hamster ovary (CHO) cells, or their derivatives such
as Veggie CHO and related cell lines which grow in serum-free media
(Rasmussen et al., 1998, Cytotechnology 28: 31), HeLa cells, baby
hamster kidney (BHK) cells (e.g., ATCC CRL 10), the CVI/EBNA cell
line derived from the African green monkey kidney cell line CVI
(ATCC CCL 70) as described by McMahan et al., 1991, EMBO J. 10:
2821, human embryonic kidney (HEK) cells such as 293, 293 EBNA or
MSR 293, human epidermal A431 cells, human Colo205 cells, HL-60
cells, U937 cells, HaK cells, Jurkat cells, HepG2/3B cells, KB
cells, NIH 3T3 cells, or S49 cells. Other mammalian cell types that
are capable of expression of a heterologous polypeptide could also
be used.
[0509] In some embodiments, an antibody mixture, e.g., a MabPair or
3-in-1 mixture, can be obtained from a population of host cells
into which DNA encoding the antibody mixture has been introduced,
for example, by transfection. In some embodiments, a single cell is
isolated from the population of cells into which the DNA has been
introduced. This cell is propagated to create a "host cell line,"
as defined herein, that can produce the antibody mixture.
Methods of Treatment
[0510] The antibodies and/or mixtures of antibodies described
herein or nucleic acids encoding them can be used to treat a
variety of diseases, optionally human diseases. As would be readily
understood by one of skill in the art, the disease that a
particular antibody or mixture of antibodies, or nucleic acids
encoding the antibody or mixture, could be used to treat could be
determined by a variety of factors including the identity of the
target protein to which each antibody in the mixture binds, the
particular epitope on each target protein bound by each antibody in
the mixture, the relative amounts of each antibody in the mixture,
the isotype of each antibody, and the in vivo half life of each
antibody in the mixture, among other possible factors. The target
proteins bound by the antibody or the antibodies in a mixture as
described herein may play a direct or indirect role in driving the
course of a disease being treated. For example, a target protein
may be part of a biological pathway that drives a disease or be a
protein that serves as an immune checkpoint, and/or a target
protein may serve as a means to target disease cells for
destruction by the immune system. Other scenarios are also
possible.
[0511] In a general sense, the mixtures of antibodies, or nucleic
acids encoding them, described herein can be used to treat diseases
driven by multiple biological pathways, diseases driven by a
molecule that has multiple mechanisms of action (e.g., HER2 in
breast cancer), or diseases driven by multiple molecules that feed
into a single biological pathway, among other possibilities. These
diseases include, without limitation, cancers, metabolic diseases,
infectious diseases, and autoimmune or inflammatory diseases, among
many possibilities.
[0512] The antibodies, mixtures of antibodies, or nucleic acids
encoding them described herein can, for example, be used to treat a
cancer. In such a case, the antibody or the mixture of antibodies
can be administered to a cancer patient, optionally directly to a
tumor. As is known in the art, different cancers are different and
require different treatments. Thus, different antibodies or
mixtures of antibodies may be suitable for different cancers.
Cancers that can be treated with the mixtures of antibodies
described herein include, for example, hematolytic cancers, solid
tumors including carcinomas and sarcomas, breast cancer, skin
cancers including melanoma, lung cancers, pancreatic cancer,
prostate cancer, cancer of the head and neck, thyroid cancer, brain
cancer, among many others.
[0513] The antibodies, the mixtures of antibodies, or the nucleic
acids encoding them can be formulated, for example, as a liquid, a
paste or a cream, or a solid. Oral administration is possible. The
antibody, antibody mixture, or nucleic acids can be administered
via parenteral injection. For example, an injection of the antibody
mixtures or nucleic acids can be subcutaneous, intravenous,
intra-arterial, intralesional (including into a tumor or other
major site of a cancer), peritoneal, or intramuscular. Topical
administration, e.g., of a liquid, paste, or cream, is possible,
especially for diseases of the skin. Administration through contact
with a mucus membrane, such as by intra-nasal, sublingual, vaginal,
or rectal administration, is also possible. Alternatively, an
antibody, antibody mixture, or nucleic acid(s) encoding an antibody
or antibody mixture can be administered as an inhalant.
[0514] In some embodiments, the nucleic acids encoding the antibody
or the mixture of antibodies can be carried on one or more viral
vectors. In such embodiments, these viral vectors containing
nucleic acids encoding the antibody or the mixture of antibodies
can be administered to patients to treat a disease, e.g., by oral
administration or by injection (including, for example,
subcutaneous, intramuscular, intravenous, intra-tumoral or
peritoneal injection), inhalation, topical administration, and/or
by administration to a mucus membrane (through which the nucleic
acids can be absorbed), among many possibilities. In a cancer
patient, such viral vectors containing nucleic acids encoding the
antibody or the mixture of antibodies can be administered directly
to a tumor or a major site of cancer cells in the patient, for
example by injection, inhalation (for a lung cancer), topical
administration (for a skin cancer), and/or administration to a
mucus membrane (through which the nucleic acids can be absorbed),
among many possibilities. The viral vector(s) can be, for example,
adenovirus, adeno-associated virus (AAV), retrovirus, vaccinia
virus, modified vaccinia virus Ankara (MVA), herpes virus,
lentivirus, or a poxvirus vector(s).
[0515] Dosing and frequency of dosing of the antibody, the mixture
of antibodies, or the nucleic acids encoding them can be adjusted
by one skilled in the art according to the condition being treated,
the concentration of the antibodies or nucleic acids, the binding
properties (such as affinity and avidity) of the antibodies, the in
vivo abundance and accessibility of the target molecules to which
the antibodies bind, and the in vivo half lives of the antibodies,
among many other possible considerations. The dose of the antibody
or mixture of antibodies administered to a patient can be, for
example, from about 0.0036 milligrams (mg) to about 450 mg, from
about 0.000051 mg/kg to about 6.4 mg/kg, or from about 0.002
mg/mm.sup.2 to about 250 mg/mm.sup.2. Similarly, dosing of nucleic
acids, e.g., DNA, encoding the antibody or antibody mixture can be,
for example, from about 10.sup.9 to about 10.sup.13 copies of the
DNA(s) encoding the antibody or antibody per kilogram of patient
weight. Dosing can occur every day, every other day, twice per
week, once per week, every other week, once every 3, 4, 5, 6, 7, 8,
9, 10, 11, or 12 weeks, 4 times per year, twice per year, once
every nine months, or once per year, among other possible
schedules.
[0516] Having described the invention in general terms above, the
specific Examples described below are offered to exemplify, not
limit, the scope of the invention. It is understood that various
changes and modifications may be made to the invention that are in
keeping with the spirit of the invention described herein and would
be apparent to one of skill in the art. Such changes and
modifications are within the scope of the invention described
herein, including in the appended claims.
EXAMPLES
Example 1: Designing HC- and LC-Partner-Directing Alterations
[0517] Preventing non-cognate HC/LC pairing would greatly limit the
number of species of antibodies produced by a host cell transfected
with DNAs encoding multiple full-length antibodies. See, e.g., FIG.
4. To ensure that only cognate LC/HC pairs form, it is critical to
find an effective way to control the kinetics of the HC/LC assembly
process so that each LC strongly favors pairing with its cognate HC
but disfavors the non-cognate HC. Since both VH/VL and CH1/CL
interfaces are involved in HC/LC recognition and engagement (see
Knarr et al. (1995), J. Biol. Chem. 270: 27589-27594; Feige et al.
(2009), Mol. Cell 34: 569-579; Reiter et al. (1996), Nat.
Biotechnol. 14: 1239-1245; Potapov et al. (2004), J. Mol. Biol.
342: 665-679; Rothlisberger et al. (2005), J. Mol. Biol. 347:
773-789, all of which are incorporated herein by reference in their
entirety), both interfaces were engineered to force cognate HC/LC
pairing as explained in detail below.
[0518] Existing X-ray crystal structures were used to identify
residues on the VH/VL and CH1/CL interfaces for modification.
Co-crystal structures of two humanized IgG1 anti-HER2 Fab fragments
complexed with HER2 have been reported, that is, the structure of
the trastuzumab Fab fragment, (available at
http://www.imgt.org/3Dstructure-DB/by searching with the Protein
Data Bank accession number 1N8Z; see also Cho et al. (2003), Nature
421(6924): 756-760, which is incorporated herein by reference) and
the structure of the pertuzumab Fab fragment, (available at
http://www.imgt.org/3Dstructure-DB/by searching with the Protein
Data Bank accession number 1578; see also Franklin et al. (2004),
Cancer Cell 5(4): 317-328, which is incorporated herein by
reference). The crystal structure of a human IgG4 Fab has also been
reported (available at http://www.imgt.org/3Dstructure-DB/by
searching with the Protein Data Bank accession number 1BBJ; see
also Brady et al. (1992), J. Mol. Biol., 227(1): 253-264, which is
incorporated herein by reference). For the purpose of selecting
residues involved in the HC/LC interaction that would be suitable
to test as sites for substitution with charged amino acids,
physical contact as determined by a distance limit criterion and
solvent accessible surface area were considered. Using the physical
contact method, interface residues for substitution with charged
amino acids were defined as residues whose side chain heavy atoms,
i.e., atoms other than hydrogen, are positioned closer than a
specified limit (5 .ANG.) from side chain heavy atoms of any
residue in the second chain. In some cases, this could mean that
the .alpha.-carbon atoms (Ca) of the two amino acids, i.e., the
carbon in the position adjacent to the carboxyl group of the amino
acid, could be as far as about 12 .ANG. away from each other. Such
distances were determined using Molecular Operating Environment
(MOE) software, obtained from Chemical Computing Group Inc. (1010
Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7). The
second method involves calculating solvent accessible surface area
(ASA) of the residues in the presence and absence of the second
chain. See, e.g., Liu et al. (2015), J. Biol. Chem. 290(12):
7535-7562, the relevant portions of which are incorporated herein
by reference. The residues that show difference>1 .ANG..sup.2 in
ASA between the two calculations were identified as interface
residues. Both the methods identified similar set of interface
residues. The following additional criteria were further applied to
select VH/VL interface residue pairs for mutagenesis: (1) they are
not in CDRs and do not make contact with any CDR residues, (2) they
are highly conserved among IgG antibody subtypes, and (3) they are
mostly solvent inaccessible (i.e., buried or partially buried).
[0519] In variable domains, G44 and Q105 in the VH domain are
spatially close to Q100 and A43 in the VL domain, respectively,
regardless of the germline sequences from which a particular
variable domain is derived. Moreover, these are solvent
inaccessible, spatially conserved residues, i.e., residues whose
spatial location within the tertiary structure of the antibody is
conserved among different antibodies. Hence these sites were chosen
for making substitutions to create charge-charge interactions
between VH and VL. We refer to such substitutions herein as "charge
pair" alterations or substitutions.
[0520] The residues at the CH1/CL interface of an IgG1 or IgG4 Fab
fragment that are close enough, i.e., less than about 12 .ANG.
between Ca atoms and about 5 .ANG. between side chain heavy atoms,
to be appropriate for charge pair substitutions are summarized in
Tables 13 and 14 below. Contacting residues are shown in the same
row, along with the distance between the .alpha.-carbons of the
residues.
TABLE-US-00014 TABLE 13 Contacting residues at IgG1 CH1/CL.kappa.
interface C.alpha.-C.alpha. CH1 distance CL.kappa. Residue
Location* EU #.sup.@ (.ANG.) EU #.sup.@ Location* Residue K
B-strand 147 9.74 124 A-strand Q K B-strand 147 10.55 129 B-strand
T K B-strand 147 9.28 131 B-strand W K B-strand 147 10.71 180
E-strand T H D-strand 168 7.33 164 D-strand T H D-strand 168 9.49
167 DE-turn D H D-strand 168 7.41 174 DE-turn S T D-strand 169 7.54
164 D-strand T Q DE-turn 175 8.29 160 D-strand Q Q DE-turn 175 9.79
180 E-strand T S DE-turn 176 10.41 160 D-strand Q S DE-turn 181
9.71 178 E-strand T S E-strand 183 7.87 176 E-strand S T E-strand
187 10.84 116 A-strand F T E-strand 187 8.88 137 B-strand N *The
locations of the various strands and turns in an immunoglobulin
structure are known in the art and illustrated in, e.g., Wang
(2013), Protein Cell. 4(8): 569-572, the relevant portions of which
are incorporated herein by reference. .sup.@Numbering is according
to Edelman et al., supra, as illustrated in Table 6 (CH1) and Table
10 (CL).
TABLE-US-00015 TABLE 14 Contacting residues at IgG4 CH1/CL.kappa.
interface C.alpha.-C.alpha. CH1 distance CL.kappa. Residue
Location* EU #.sup.@ (.ANG.) EU #.sup.@ Location* Residue R
A-strand 133 11.51 117 A-strand I E B-strand 137 8.30 114 A-strand
S E B-strand 137 9.94 116 A-strand F K B-strand 147 9.65 124
A-strand Q K B-strand 147 10.83 129 B-strand T K B-strand 147 9.44
131 B-strand S K B-strand 147 10.96 178 E-strand T K B-strand 147
11.39 180 E-strand T H D-strand 168 7.37 164 D-strand T H D-strand
168 9.52 167 DE-turn D H D-strand 168 9.71 173 DE-turn Y H D-strand
168 7.66 174 DE-turn S F D-strand 170 7.41 176 E-strand S V
D-strand 173 7.68 160 D-strand Q V D-strand 173 6.66 162 D-strand S
Q DE-turn 175 8.16 160 D-strand Q Q DE-turn 175 9.61 180 E-strand T
S DE-turn 181 9.32 180 E-strand T S E-strand 183 7.61 176 E-strand
S *The locations of the various strands and turns in an
immunoglobulin structure are illustrated in, e.g., Wang (2013),
Protein Cell. 4(8): 569-572. .sup.@Numbering is according to
Edelman et al., supra, as illustrated in Table 6 (CH1) and Table 10
(CL).
[0521] Due to the presence of the CH1 domain, HCs are retained in
the endoplasmic reticulum (ER) by immunoglobulin heavy chain
binding protein (BiP, also known as GRP78 and HSPA5) until they are
engaged by LCs for the assembly of a full-length IgG. BiP generally
recognizes and binds to hydrophobic residues and, to lesser extent,
to hydrophilic and charged residues. Knarr et al. (1995), J Biol
Chem 270: 27589-27594. Hence, destabilization of HC/LC interactions
could cause an antibody to be retained in the ER, thus reducing
secretion of the antibody into culture medium.
[0522] Examination of the VH/VL and CH1/CL interface tertiary
structures revealed that hydrogen bonds and van der Waals
interactions are important parts of these interfaces. Unlike the
CH3/CH3 interface, electrostatic charge-charge residue interactions
are rare between the LC and HC. For example, a CL.kappa./CH1
interface has one and a CL.lamda./CH1 interface has two
positive-negative charge interactions involving E/D and K residues.
All of these existing charge pair interactions involve partially or
fully solvent exposed positions, a situation that weakens an
electrostatic interaction due to interference of solvent
molecules.
[0523] To utilize an electrostatic steering effect to drive cognate
HC/LC pairing, we hypothesized that it may be important to keep the
VH/VL and CH1/CL interfaces stable and that residue substitutions
could destabilize these structures, thereby decreasing antibody
expression. Therefore, we preferentially used at least one
conserved, naturally occurring charged residue (for example, K147
and H168 in the CH1 domain of both IgG1 and IgG4) for exploration.
We hypothesized that changing a conserved charged residue in the
CH1 domain to a residue having the opposite charge might cause
little or no disruption of the CH1/CL interface.
[0524] Our analysis of the tertiary structures described above
indicated that K147 in the CH1 domains of both IgG1 and IgG4
contacts Q124, T129, S131, and T180 in a CL.kappa. domain and H168
in a CH1 domain of both IgG1 and IgG4 contacts T164, D167, and S174
in a CL.kappa. domain. We hypothesized that an oppositely charged
pair of amino acids interacting across a CL/CH1 interface might
have maximum electrostatic steering effect if one of the oppositely
charged residues is located on the inner .beta.-sheet of the CL
and/or CH1 domain, which includes Strand-B, Strand D, and Strand E.
Examples of such residues include S131, T180, T164, and S174 on the
inner .beta.-sheet of a CL.kappa. domain. We focused on (1)
substitutions at S131 and/or T180 in a CL.kappa. domain that would
promote an electrostatic interaction with the residue at position
147 in a CH1 domain (which may or may not have a substitution), (2)
substitutions at T164 and/or S174 in a CL.kappa. domain that would
promote an electrostatic interaction with the residue at 168 in a
CH1 domain (which may or may not have a substitution).
Example 2: Designing and Testing Added Disulfide Bridges in the
HC/LC Interface
[0525] We hypothesized that alterations that create additional
HC/LC interchain disulfide bridges might strengthen the association
of cognate HC/LC pairs. In unmodified antibodies, interchain
disulfide bonds are solvent exposed while intrachain disulfide
bonds are buried between the two layers of anti-parallel
.beta.-sheet structures within each domain, i.e., not solvent
exposed. For example, the interchain disulfide bonds in the hinge
region are solvent exposed, as is the disulfide bond between the HC
and the LC. Solvent exposed cysteine residues are considered more
reactive than non-exposed cysteine residues. Hence, in making
cysteine substitutions, we tried to create disulfide bridges that
were partially or fully solvent exposed.
[0526] Introduced disulfide bridges have been shown to stabilize Fv
fragments by stabilizing VHNL interactions, leading to higher
production and solubility of the resulting Fv fragments. Reiter et
al. (1996), Nature Biotechnol. 14(10): 1239-1245. The altered
residues for these cysteine substitutions were well-conserved amino
acids in framework regions, specifically VH residues G44 and Q105
and the contacting VL residues Q100 and A43, respectively.
[0527] We have tried a similar approach in the CH1 and CL domains.
Our analysis of the tertiary structure described in Example 1
indicates that the following residue pairs located at the CH1/CL
interface of human IgG1 or IgG4 HC with a .kappa.LC are close
enough to potentially form a disulfide bond if substituted with
cysteine.
TABLE-US-00016 TABLE 15 IgG1 CH1/CL.kappa. interface residues
suitable for cysteine substitution C.alpha.-C.alpha. IgG1 CH1
distance CL.kappa. EU #.sup.@ Location* Residue (.ANG.) Residue
Location* EU #.sup.@ 126 A-strand F 6.41 S A-strand 121 126
A-strand F 6.41 Q A-strand 124 128 A-strand L 6.03 F A-strand 118
133 A-strand K 5.52 I A-strand 117 133 A-strand K 7.09 F G-strand
209 134 A-strand S 4.62 F A-strand 116 141 B-strand A 7.49 F
A-strand 116 168 D-strand H 7.40 S DE-turn 174 170 D-strand F 6.11
S D-strand 162 170 D-strand F 7.42 S E-strand 176 173 D-strand V
7.39 Q D-strand 160 183 E-strand S 7.66 S E-strand 176 *The
locations of the various strands and turns in an immunoglobulin
structure are illustrated in, e.g., Wang (2013), Protein Cell.
4(8): 569-572. .sup.@Numbering is according to Edelman et al.,
supra, as illustrated in Table 6 (CH1) and Table 10 (CL).
TABLE-US-00017 TABLE 16 IgG4 CH1/CL.kappa. interface residues
suitable for cysteine substitution C.alpha.-C.alpha. IgG4 CH1
distance CLk EU #.sup.@ Location* Residue (.ANG.) Residue Location*
EU #.sup.@ 126 A-strand F 6.76 S A-strand 121 126 A-strand F 6.47 Q
A-strand 124 127 A-strand P 6.36 S A-strand 121 128 A-strand L 6.00
F A-strand 118 141 B-strand A 7.31 F A-strand 116 168 D-strand H
7.66 S DE-turn 174 170 D-strand F 6.32 S D-strand 162 173 D-strand
V 6.80 S D-strand 162 *The locations of the various strands and
turns in an immunoglobulin structure are illustrated in, e.g., Wang
(2013), Protein Cell. 4(8): 569-572. .sup.@Numbering is according
to Edelman et al., supra, as illustrated in Table 6 (CH1) and Table
10 (CL).
[0528] To determine whether antibodies having cysteine
substitutions in their HCs and LCs at one or more pairs of sites
set forth in Table 15 and 16 and lacking the disulfide bridge
normally present between an HC and LC would form HC/LC pairs
sufficiently stable for escape from the ER, DNA constructs encoding
such antibodies were made as follows. A DNA fragment encoding a
signal peptide (SP) followed by VH and CH1 domains from an
anti-CTLA4 antibody 1E1 (the "1E1 antibody") was synthesized by
Integrated DNA Technologies (IDT), Inc. (Iowa, USA). The amino acid
sequence of the VH and CH1 domains of the 1E1 antibody are shown in
amino acids 1-216 of SEQ ID NO:38. It was fused by Gibson reaction
(see, e.g., Gibson Assembly.RTM. Master Mix Instruction Manual, New
England Biolabs Inc. (NEB), Version 3.3, NEB catalog no. #E2611S/L,
NEB Inc. Ipswich, Mass., USA) with a downstream DNA fragment
encoding the hinge, CH2, and CH3 domains of a human IgG1 antibody
in a mammalian expression vector. The CH3 domain included the
substitutions D399K and K409E, which are discussed in more detail
in Example 4.
[0529] A second DNA fragment encoding an SP followed by VL and CL
domains from the 1E1 antibody was synthesized by IDT, Inc. and
fused by Gibson reaction with a mammalian expression vector. The
amino acid sequence of the VL and CL domains of the 1E1 antibody
are shown in amino acids 1-214 of SEQ ID NO:40. The reaction
mixture was transformed into competent E. coli XL1 Blue by
electroporation and plated out onto the LB-agar plates containing
antibiotic carbernicillin. Resulting colonies were picked and
cultured, and plasmid DNA was isolated. The plasmid insert sequence
was confirmed by DNA sequencing.
[0530] Mutations encoding amino acid substitutions were introduced
into these DNA constructs by mutagenesis reactions with QuikChange
Lightning multi site-directed mutagenesis kit (Agilent
Technologies, cat no. 210516). In the DNA encoding the HC,
mutations encoding a C220S alteration (which is replacement of the
cysteine at position 221 in SEQ ID NO:38 with serine, since the EU
numbering system is used herein to describe constant domain
alterations) were made. Mutations encoding a C214S alteration
(which is replacement of the cysteine at position 214 in SEQ ID
NO:40 with serine) were made in the DNA encoding the LC. Together,
these alterations completely eliminate the naturally occurring
interchain disulfide bridge between the cysteine residues normally
present at these positions. Further, DNA encoding the HC was
mutated to encode the alteration F126C, L128C, S134C, H168C, K133C,
F170C, V173C, or S183C. Positions 126, 128, 134, 168, 133, 170,
173, and 183 according to the EU numbering scheme are equivalent to
positions 127, 129, 135, 169, 134, 171, 174, and 184 in SEQ ID
NO:38, respectively. Similarly, DNA encoding the LC was mutated to
encode the alteration F116C, I117C, F118C, Q124C, Q160C, S162C,
S174C, S176C, or F209C. Positions 116, 117, 118, 124, 160, 162,
174, 176, and 209 according to the EU numbering are equivalent to
these same positions in SEQ ID NO:40
[0531] A second set of DNA constructs encoding altered versions of
an anti-PD1 antibody was also made in a similar manner. A DNA
fragment encoding an SP followed by a VH domain and an IgG4 CH1
domain from the anti-PD1 102 antibody were synthesized by IDT, Inc.
and fused by Gibson reaction with a downstream DNA fragment
encoding the hinge, CH2, and CH3 domains of a human IgG4 antibody
in a mammalian expression vector. SEQ ID NO:23 provides the amino
acid sequence of the HC of the anti-PD1 antibody 16137, which
differs from that of the anti-PD1 102 antibody HC only in the CDRs
and at three additional amino acids in the framework regions of the
VH domain. Like the anti-PD1 102 antibody HC, the anti-PD1 antibody
16137 HC has a proline at position 228 (using EU numbering) to
prevent Fab arm exchange (Silva et al. (2015), J. Biol. Chem.
290(9): 5462-5469, which is incorporated herein by reference)). A
second DNA fragment encoding an SP followed by VL and CL domains
from the anti-PD1 102 antibody was synthesized by IDT, Inc. and
fused by Gibson reaction with a mammalian expression vector. SEQ ID
NO:24 provides the amino acid sequence of the LC of the anti-PD1
antibody 16137, which differs from that of the anti-PD1 102
antibody LC only in the CDRs and at five additional amino acids in
the framework regions of the VL domain. The reaction mixture was
transformed into competent E. coli XL1 Blue by electroporation and
plated out onto the LB-agar plates containing antibiotic
carbernicillin. The resulting colonies were picked and cultured,
and the insert sequence of the isolated plasmid DNA was confirmed
by DNA sequencing.
[0532] Amino acid substitutions were introduced into the anti-PD1
antibody using a QuikChange Lightning multi site-directed
mutagenesis kit (Agilent Technologies, cat no. 210516). To
eliminate the disulfide bridge between C131 (HC) and C214 (LC),
mutations in the HC and LC DNA constructs encoding antibodies with
the alterations C131S (HC) and C214S (LC) were made. Further, DNA
constructs encoding antibodies with the HC alteration F126C, P127C,
L128C, H168C, F170C, V173C, or S183C and the LC alteration F118C,
S121C, Q124C, S162C, S174C, or S176C were made. High quality
plasmid DNAs (0D260/280=1.90-2.00) were prepared by using Qiagen
Midi-prep kit. The plasmid DNAs were diluted in water and mixed in
EPPENDORF TUBES.RTM..
[0533] All of the altered antibodies described immediately above
were missing the disulfide bridge normally present between C131
(IgG4 HC) or C220 (IgG1 HC) and C214 (LC) but have an additional
pair of cysteine residues in the HC and LC that could potentially
form a disulfide bridge. Two DNAs encoding an HC/LC pair were
combined to transfect EXPI293.TM. cells (ThermoFisher Scientific
Inc., Waltham, Mass., USA), and the antibodies secreted by the
transfectants into the culture medium were visualized by Western
blotting. If the transfectants produced full-length antibody of
about 150 kilodaltons (kDa) in size, disulfide bridge formation was
judged to be successful.
[0534] In more detail, the EXPI293.TM. cells were transfected with
the plasmid DNAs encoding the test antibody with LIPOFECTAMINE.RTM.
2000 in 24-well deep well blocks. Cells were continuously shaken at
150 revolutions per minute (rpm) at 37.degree. C. for 4 days. The
supernatants were harvested by spinning down cells at 1500 rpm for
20 minutes. For non-reduced samples, 5 microliters (.mu.l) of
supernatant and 5 .mu.l of 2.times. Laemmli Sample Buffer (65.8 mM
Tris-HC1, pH 6.8, 2.1% sodium lauryl sulfate (SDS), 26.3% (w/v)
glycerol, 0.01% bromophenol blue) were heated at 70.degree. C. for
10 minutes. For the reduced samples, 5 .mu.l of supernatant and 5
.mu.l of 2.times. Laemmli Sample Buffer in the presence of 100 mM
dithiothreitol (DTT) were heated at 70.degree. C. for 10 minutes.
In the Western blots shown in FIGS. 5 and 6, all samples were
non-reduced. The treated samples were loaded into the wells of
4-15% CRITERION.TM. TGX STAIN-FREE.TM. Precast SDS-PAGE gels
(Bio-Rad Laboratories, Inc., Hercules, Calif., cat no. 567-8085).
Electrophoresis was run for 45 minutes at 200 V. The proteins were
transferred onto a nitrocellulose membrane with TRANS-BLOT.RTM.
TURBO.TM. Transfer System (Bio-Rad Laboratories, Inc.) and blocked
in 3% non-fat milk in 1.times. phosphate buffered saline with 0.05%
TWEEN.RTM. 20 (PBST). The nitrocellulose membrane was washed, and
the antibodies were detected with HRP-conjugated polyclonal
goat-anti-human IgG (Fc-specific) (Sigma-Aldrich Corporation, St.
Louis, Mo., cat. no. A0170). The image was visualized with a
CHEMIDOC.TM. XRS+ imager from Bio-Rad Laboratories, Inc.
[0535] Results are shown in FIGS. 5 and 6. As explained in Example
1, HCs are generally retained in the ER unless they are engaged by
LCs. Hence, if the introduced cysteine residues were forming
disulfide bridges, it would be expected that the HCs would be
engaged by the LCs and that the transfectants would therefore
produce primarily full-length antibody of about 150 kDa, indicated
by the top arrow in FIG. 5 and the 150 kDa size marker in FIG. 6.
However, other species could possibly be produced, such as a
half-antibody consisting of 1 HC and 1 LC (75 kDa; bottom arrow in
FIG. 5), a three quarters antibody containing 2 identical HCs and 1
LC (125 kDa; second arrow from top in FIG. 5) and 2 HCs (100 kDa;
third arrow from top in FIG. 5 and the 100 kDa size marker in FIG.
6). Such species might be produced in situations where the
introduced cysteine residues are not completely effective in
forming a disulfide bridge.
[0536] It was expected that antibodies lacking the cysteine
residues normally present at C131 (IgG4 HC) or C220 (IgG1 HC) and
C214 (LC) (which form the naturally-occurring interchain disulfide
bridge) would not form full-length antibodies unless both the HC
and LC were substituted with cysteine at contacting sites. Data
using antibodies lacking the naturally-occurring disulfide bridge
and not containing any cysteine substitutions are shown in FIG. 6,
lanes 2 and 8. Only 100 kDa species (likely HC/HC) were detected.
Results using antibodies lacking the naturally-occurring disulfide
bridge and containing a cysteine substitution in their HC, but not
in their LC, or vice versa, are shown in lanes 14-22 and lanes
25-33 of FIG. 5. As expected, most of these HC/LC combinations did
not produce significant quantities of full-length antibody,
producing mostly a 100 kDa species (likely HC/HC). Surprisingly,
three of these HC/LC combinations formed full-length antibody,
i.e., K133C in the CH1 domain and no substitution in the LC (FIG.
5, lane 15) and I117C or F209C in the LC and no substitution in the
HC CH1 domain (FIG. 5, lanes 26 and 27, respectively). Lanes 2-12
of FIG. 5 show data from HC/LC combinations where both the HC and
the LC contained cysteine substitutions at contacting sites as
indicated in the description of FIG. 5 and lacked the cysteines
that form the naturally-occurring disulfide bridge. All but two of
these HC/LC combinations (both of which contained S183C (HC) plus
S176C (LC) (lanes 8 and 12)) formed significant quantities of
full-length antibody. Similarly, analysis of further samples
lacking the naturally-occurring disulfide bridge and comprising
cysteine substitutions at contacting residues detected mostly
full-length antibody. FIG. 6, lanes 3-6 and 9-11.
[0537] Thus, these data suggest that most, but not all, of the
introduced pairs of cysteine residues did form disulfide bridges,
although the presence of detectable quantities of
smaller-than-full-length antibody species suggests that kinetics of
antibody secretion may be affected by the introduced cysteine
residues. Specifically, the results suggest that in a human IgG1
antibody, cysteine substitutions F126C (CH1)-Q124C (CL.kappa.),
L128C (CH1)-F118C (CL.kappa.), S134C (CH1)-F116C (CL.kappa.), H168C
(CH1)-S174C (CL.kappa.), K133C (CH1)-I117C (CL.kappa.), K133C
(CH1)-F209C (CL.kappa.), V173C (CH1)-Q160C (CL.kappa.), F170C
(CH1)-S162C (CL.kappa.), and F170C (CH1)-S176C (CL.kappa.) can
mediate disulfide bond formation. Further, the results suggest that
in a human IgG4 antibody, cysteine substitutions F126C (CH1)-S121C
(C.kappa.), F126C (CH1)-Q124C (C.kappa.), P127C (CH1)-S121C
(C.kappa.), L128C (CH1)-F118C (C.kappa.), H168C (CH1)-S174C
(C.kappa.), V173C (CH1)-S162C (C.kappa.), and F170C (CH1)-S162C
(C.kappa.) can mediate disulfide bond formation.
Example 3: Testing Antibody Mixtures Containing LC- and
HC-Partner-Directing Alterations that are Charge Pair Substitutions
and/or Cysteine Substitutions
[0538] Having determined through analysis of published tertiary
structures the identity of a number of contacting pairs of residues
in the HC and LC suitable for making charge-pair or cysteine
substitutions (Examples 1 and 2) and having made and tested some
cysteine substitutions, a number of such substitutions and
combinations thereof were made and tested to determine their
effects on cognate HC/LC pairing. To quickly assess the efficacy of
various LC- and HC-partner-directing alterations in forcing cognate
HC/LC pairing, "chain drop out" experiments as described below were
performed.
[0539] DNA constructs encoding the antibodies were made using
methods similar to those described in Example 2. A DNA fragment
encoding the human IgG1 HC of the human anti-CTLA4 111 antibody
(the "111 antibody") was made as described for the HC of the
anti-CTLA4 1E1 antibody in Example 2. The CH1, hinge, CH2 and CH3
domains and the framework regions of the VH domain of the 111
antibody have the same amino acid sequences as those of the HC of
the 1E1 antibody. The amino acid sequence of the HC of the 1E1
antibody is provided in SEQ ID NO:38. Another DNA fragment encoding
an SP and the VL and CL.kappa. domains of the LC of the human
anti-CTLA4 111 antibody was also made as described for the
anti-CTLA4 1E1 antibody in Example 2. The CL domain and the
framework regions of the VL domain of the 111 antibody have the
same amino acid sequence as those of the LC of the 1E1 antibody,
the sequence of which is provided in SEQ ID NO:40.
[0540] The construction of vectors containing DNA inserts encoding
the HC and LC of the anti-PD1 102 antibody are described in Example
2.
[0541] Similar constructions were made to obtain DNA encoding a
pair of anti-HER2 antibodies called 4D5-8 and 2C4. The antibody
4D5-8 contains the variable regions from the antibody humAb4D5-8
described in Carter et al. (1992), Proc. Natl. Acad. Sci. USA
89:4285-4289, which is incorporated herein in its entirety, and
constant regions from a human IgG1 HC and a human .kappa.LC. The
antibody 2C4 contains the variable regions from the antibody rhuMAb
2C4 described in Adams et al. (2006), Cancer Immunol. Immunother.
55(6): 717-727, which is incorporated herein in its entirety, and
constant regions from a human IgG1 HC and a human .kappa.LC. The
amino acid sequences of the light and heavy chains of 4D5-8 are
provided in SEQ ID NOs: 19 and 20, respectively. The amino acid
sequences of the heavy and light chains of 2C4 are provided in SEQ
ID NOs: 21 and 22, respectively.
[0542] Amino acid substitutions were introduced into the DNAs
encoding the anti-CTLA4 and anti-PD1 antibodies or the two
anti-HER2 antibodies by mutagenesis reactions with QuikChange
Lightning multi site-directed mutagenesis kit (Agilent
Technologies, Santa Clara, Calif., cat no. 210516). Each QuikChange
process resulted in a vector DNA containing an insert comprising
the desired mutation(s), which was then transformed into E. coli
XL1-Blue cells. Colonies from these transformations were picked and
cultured to obtain plasmid DNA for transfection into mammalian
cells. Sequences of all constructs were confirmed by DNA
sequencing.
[0543] The plasmid DNAs from these cultured colonies were purified
using a Qiagen.RTM. Midi-prep kit (Qiagen N.V., the Netherlands).
The resulting DNAs were diluted in water and mixed in EPPENDORF
TUBES.RTM.. For each variant pair, a set of 5 tubes of mixed DNAs
were transiently transfected into EXPI293.TM. cells (ThermoFisher
Scientific Inc.) to monitor the fidelity of HC/LC pairings. Tube 1
contained DNAs encoding both full-length antibodies, e.g., DNA
encoding the anti-PD1 HC and LC (HC1 and LC1) and the anti-CTLA4 HC
and LC (HC2 and LC2)). Tube 2 contained DNAs encoding only one
antibody (HC1 and LC1). Tube 3 contained the DNAs encoding a
non-cognate HC/LC pair (HC1 and LC2). Tube 4 contained DNAs
encoding only one antibody (HC2 and LC2). Tube 5 contained DNAs
encoding a non-cognate HC/LC pair (HC2 and LC1). DNAs encoding
unrelated antibodies were transfected in parallel to assess
transfection efficiency.
[0544] The mammalian EXPI293.TM. cells were transfected with the
DNAs described above using LIPOFECTAMINE.RTM. 2000 (ThermoFisher
Scientific, Waltham, Mass., USA) in 24-well deep well plates. Cells
were continuously shaken at 150 rpm at 37.degree. C. for 4 days.
The supernatants were harvested for analysis by spinning down cells
at 1500 rpm for 20 min. Samples were prepared and subjected to
electrophoresis, the gels were blotted, the nitrocellulose
membranes were blocked and washed, and the antibodies were detected
as explained in Example 2.
[0545] The mammalian EXPI293.TM. cells used for transfection can
transcribe DNAs, translate mRNAs and secret antibodies into the
culture medium. Western blotting of non-reduced samples of
antibodies in the culture media was carried out to monitor the
HC/LC pairings. As explained above, there were five
co-transfections using the five different DNA mixtures described
above for each pair of variants. As an initial criterion (see Table
17), a particular pair of variants was judged to be successful if
the levels of full-length antibody produced by transfectants
containing DNAs encoding cognate HC/LC pairs was higher than the
levels produced by transfectants containing only DNAs encoding
non-cognate HC/LC pairs, i.e., Tubes 3 and 5 described above.
Ultimately (see Tables 20 and 21), a particular variant mixture was
judged to be successful in forcing mostly or only cognate HC/LC
pairing if the samples from transfectants containing DNA encoding
LC1 and HC1 (Tube 2 described above), LC2 and HC2 (Tube 4), and
LC1, HC1, LC2, and HC2 (Tube 1) all produced good levels of
full-length antibody, whereas the samples from transfectants
containing DNA encoding HC1 and LC2 (Tube 3 described above) and
HC2 and LC1 (Tube 5) produced little or no detectable full-length
antibody.
[0546] Examples of such Western blots are shown in FIGS. 7-10. In
FIG. 7, lanes 21 and 22 show samples from transfectants that
received DNA encoding the anti-HER2 antibodies 4D5-8 and 2C4,
respectively, which are full-length antibodies included as controls
for transfection efficiency. The DNA contents of the test
transfectants are indicated below lanes 1-20 of FIG. 7, and the
name of the pair of variants tested is indicated above. The
identity of the alterations in the pairs of variants is stated in
Table 21 below. The results shown in FIG. 7 indicate that the
variant mixtures 18B and 18C were more effective at forcing cognate
HC/LC pairing than mixtures 18A and 18D. Transfectants containing
only non-cognate 18B or 18C HC/LC pairs produced little or no
detectable full-length antibody (FIG. 7, lanes 8, 10 13, and 15),
while transfectants containing cognate 18B or 18C HC/LC pairs
produced detectable full-length antibody (FIG. 7, lanes 6, 7, 9,
11, 12, and 14). In contrast, 18A Tube 1 and Tube 4 transfectants,
which contain DNA encoding cognate HC/LC pairs produced relatively
low amounts of full-length antibody, which was diffuse in size in
Tube 4 transfectants. FIG. 7, lanes 1 and 4. Further, 18D
transfectants containing DNA encoding only one non-cognate HC/LC
pair produced low amounts of full-length antibody (FIG. 7, lane
18), while tranfectants containing DNAs encoding cognate HC/LC
pairs produced only relatively low amounts of full-length antibody
(FIG. 7, lanes 16, 17, and 19).
[0547] Similarly, a Western blot containing data for variant
mixtures 14A to 14D (see Table 20) is shown in FIG. 8. Variants 14C
and 14D showed only cognate HC/LC pairing since there was no
detectable antibody expression when only non-cognate HC/LC pairs
(LC1+HC2 or LC2+HC1) were combined (FIG. 8, lanes 13, 15, 18, and
20), whereas cognate HC/LC pairs (LC1+HC1 or LC2+HC2) or a mixture
of all four chains (LC1+HC1+LC2+HC2) produced significant amounts
of full-length antibodies (FIG. 8, lanes 11, 12, 14, 16, 17, and
19). In contrast, mixtures 14A and 14B did show detectable
full-length antibody in some lanes containing samples from
transfectants containing only non-cognate HC/LC pair (FIG. 8, lanes
5 and 10).
[0548] In FIG. 9, similar data for variant pairs 23A-23D are shown.
The alterations in these pairs of anti-PD1 and anti-CTLA4
antibodies are shown in Table 21. As indicated under lanes 1-20,
for each pair of variants a set of five DNA combinations (as
described above) was transfected into the host cells. DNAs encoding
the anti-PD1 antibody (lanes labeled "a-PD1") and anti-CTLA4
antibody (lanes labeled "a-CTLA4") were transfected at the same
time to monitor transfection efficiency. Host cells receiving DNA
encoding antibody chains of variant pair 23A produced little
full-length antibody, regardless of whether the cells were
transfected with DNAs encoding cognate or non-cognate HC/LC pairs.
FIG. 9, lanes 1-5. Host cells receiving DNA encoding chains of
variant pairs 23B and 23C produced little or no full-length
antibody when DNAs encoding the cognate pair HC1 and LC1 were
introduced into the host cells (FIG. 9, lanes 7 and 12) and small
but detectable amounts of full-length antibody in the presence of
DNAs encoding the non-cognate pair HC2 and LC1 (FIG. 9, lanes 10
and 15). In contrast, host cells receiving DNAs encoding antibody
chains of variant pair 23D produced significant amounts of
full-length antibody when DNAs encoding cognate HC/LC pairs were
present (FIG. 9, lanes 16, 17, and 19) and little or no full-length
antibody when only DNAs encoding non-cognate HC/LC pairs were
present (FIG. 9, lanes 18 and 20).
[0549] Many pairs of variants were tested in similar chain drop out
experiments, starting with alterations introducing charged amino
acids only in the VH and VL domains or only in the CH1 and CL
domains and then combining alterations in VH, CH1, VL, and CL. In
some cases alterations creating additional disulfide bridges were
added. The identity of such pairs of alterations and the results of
the Western blots are catalogued in Tables 17-21.
TABLE-US-00018 TABLE 17 Charge pair alterations tested in chain
drop out experiments Accurate anti-HER2 4D5-8 anti-HER2 2C4 HC/LC
HC1 LC1 HC2 LC2 partner Variant.sup.# VH CH1 VL CL VH CH1 VL CL
selection* 1A G44E A43R G44R A43E N Q105E Q100R Q105R Q100E 1B G44E
A43E G44R A43R Y Q105R Q100R Q105E Q100E 1C G44R A43R G44E A43E N
Q105E Q100E Q105R Q100R 1D G44R A43E G44E A43R N Q105R Q100E Q105E
Q100R 2A K147E S131R K147R S131E N 2B K147E T180R K147R T180E N 2C
K147R S131E K147E S131R Y 2D K147R T180E K147E T180R N 3A H168E
T164R H168R T164E N 3B H168E S174R H168R S174E Y 3C H168E S174R
H168R S174D Y 3D H168R T164E H168E T164R N 4A H168R S174E H168E
S174R N 4B H168R S174D H168E S174R N 4C S181E T178R S181R T178E N
4D S181R T178E S181E T178R Y .sup.#This column provides the
designation for the particular pair of altered variant antibodies.
*A "Y" indicates pairs of variants where transfectants containing
DNAs encoding cognate HC/LC pairs had robust expression of
full-length antibody and produced more full-length antibody than
transfectants containing only DNAs encoding non-cognate HC/LC
pairs. Such rows are shown in boldface. An "N" indicates that this
criterion was not met.
[0550] The data in Table 17 indicate that certain alterations in
the VH and VL and in the CH1 and CL were more effective than others
at forcing cognate HC/LC pairings. The 1B pair of variants was
chosen as a starting point for further experiments to test various
combinations of alterations introducing charged amino acids in the
VH, CH1, VL, and CL domains as reported in Table 18 below.
TABLE-US-00019 TABLE 18 Charge pair alterations tested in chain
drop out experiments Accurate anti-HER2 4D5-8 anti-HER2 2C4 HC/LC
HC1 LC1 HC2 LC2 partner Variant.sup.# VH CH1 VL CL VH CH1 VL CL
selection* 5A G44E K147R A43E S131D G44R K147D A43R S131R N Q105R
Q100R Q105E Q100E 5B G44E K147D A43E S131R G44R K147R A43R S131D N
Q105R Q100R Q105E Q100E 5C G44E H168R A43E S174D G44R H168D A43R
S174R N Q105R Q100R Q105E Q100E 5D G44E H168D A43E S174R G44R H168R
A43R S174D N Q105R Q100R Q105E Q100E 6A G44E S181R A43E T178D G44R
S181D A43R T178R N Q105R Q100R Q105E Q100E 6B G44E S181D A43E T178R
G44R S181R A43R T178D N Q105R Q100R Q105E Q100E 6C G44E K147R A43E
S131D G44R K147D A43R S131R Y Q105R H168R Q100R S174D Q105E H168D
Q100E S174R 6D G44E K147R A43E S131D G44R K147D A43R S131R N Q105R
H168D Q100R S174R Q105E H168R Q100E S174D 7A G44E K147D A43E S131R
G44R K147R A43R S131D N Q105R H168R Q100R S174D Q105E H168D Q100E
S174R 7B G44E K147D A43E S131R G44R K147R A43R S131D Y Q105R H168D
Q100R S174R Q105E H168R Q100E S174D 7C G44E K147R A43E S131E G44R
K147E A43R S131R N Q105R Q100R Q105E Q100E 7D G44E A43E G44R A43R N
Q105R Q100R Q105E Q100E 8A G44E K147R A43E S131E G44R K147E A43R
S131R N Q105R Q100R Q105E Q100E 8B G44E K147E A43E S131R G44R K147R
A43R S131E N Q105R Q100R Q105E Q100E 8C G44E H168R A43E S174E G44R
H168E A43R S174R N Q105R Q100R Q105E Q100E 8D G44E H168E A43E S174R
G44R H168R A43R S174E N Q105R Q100R Q105E Q100E 9A G44E S181R A43E
T178E G44R S181E A43R T178R N Q105R Q100R Q105E Q100E 9B G44E S181E
A43E T178R G44R S181R A43R T178E N Q105R Q100R Q105E Q100E 9C G44E
K147R A43E S131D G44R K147D A43R S131R N Q105R H168R Q100R S174D
Q105E H168D Q100E S174R 9D G44E K147D A43E S131R G44R K147R A43R
S131D N Q105R H168D Q100R S174R Q105E H168R Q100E S174D *A "Y"
indicates pairs of variants where transfectants containing DNAs
encoding cognate HC/LC pairs were more clearly distinguished from
transfectants containing DNAs encoding only non-cognate HC/LC pairs
(with the former showing expression of full-length antibody and the
latter showing less or no expression) than was observed in the 1B
pair of variants. Such rows are shown in boldface. A "N" indicates
that this criterion was not met. .sup.#This column provides the
designation for the particular pair of altered antibodies.
[0551] Starting with the alterations present in the 6C and 7B pairs
of variants, all glutamic acid residues that had been introduced
were changed to aspartic acid to determine whether these changes
would have an effect on the selectivity of HC/LC pairing. These
alterations and their effects are cataloged in Table 19.
TABLE-US-00020 TABLE 19 E to D substitutions tested in chain drop
out experiments Accurate anti-HER2 4D5-8 anti-HER2 2C4 HC/LC HC1
LC1 HC2 LC2 partner Variant.sup.# VH CH1 VL CL VH CH1 VL CL
selection* 10A G44E K147R A43E S131D G44R K147D A43R S131R N Q105R
H168R Q100R S174D Q105E H168D Q100E S174R 10B G44D K147R A43D S131D
G44R K147D A43R S131R Y Q105R H168R Q100R S174D Q105D H168D Q100D
S174R 10C G44E K147R A43E Q124D G44R K147D A43R Q124R N Q105R H168R
Q100R S174D Q105E H168D Q100E S174R 11A G44E K147D A43E S131R G44R
K147R A43R S131D N Q105R H168D Q100R S174R Q105E H168R Q100E S174D
11B G44D K147D A43D S131R G44R K147R A43R S131D Y Q105R H168D Q100R
S174R Q105D H168R Q100D S174D 11C G44E K147D A43E Q124R G44R K147R
A43R Q124D N Q105R H168D Q100R S174R Q105E H168R Q100E S174D *A "Y"
indicates pairs of variants where transfectants containing DNAs
encoding cognate HC/LC pairs were more clearly distinguished from
transfectants containing DNAs encoding only non-cognate HC/LC pairs
(with the former showing expression of full-length antibody and the
latter showing less or no expression) than was observed in the 6C
and 7B pair of variants. Such rows are shown in boldface. A "N"
indicates that this criterion was not met. .sup.#This column
provides the designation for the particular pair of altered variant
antibodies.
[0552] The results in Table 19 indicate that the substitution of D
for E in pairs of variants further strengthened cognate HC/LC
pairing as compared to non-cognate HC/LC pairing. We hypothesize
that the smaller molecular size of D as compared to E may be less
disruptive to interactions across the VHNL and CH1/CL
interfaces.
[0553] Starting from the alterations present in the 10B pair of
variants, we made further alterations that were designed to create
an additional disulfide bridge between the HC and the LC. We hoped
that such additional disulfide bridges would increase antibody
expression from transfectants containing DNAs encoding cognate
HC/LC pairs, many of which were not expressing the desired high
levels antibody. We hypothesized that the charge pair alterations
were somehow destabilizing cognate HC/LC pairs, causing retention
of the antibodies in the ER, and that an additional disulfide
bridge might stabilize the cognate pairs, thereby increasing
secretion of the antibodies into the culture medium where they
could be detected. As reported in Example 2, these cysteine
substitutions also have been tested in the absence of charge pair
substitutions to ascertain their ability to form HC/LC disulfide
bonds. These combinations of alterations and their effects are
cataloged in Table 20 below.
TABLE-US-00021 TABLE 20 Charge pairs plus cysteine pairs tested in
chain drop out experiments Accurate anti-HER2 4D5-8 anti-HER2 2C4
HC/LC HC1 LC1 HC2 LC2 partner Variant.sup.# VH CH1 VL CL VH CH1 VL
CL selection* 12A G44D K133C A43D F209C G44R F170C A43R S162C N
Q105R K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D H168D
S174R 12B G44D K133C A43D F209C G44R V173C A43R+ Q160C N Q105R
K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D H168D S174R
12C G44D K133C A43D F209C G44R F170C A43R+ S176C N Q105R K147R
Q100R S131D Q105D K147D Q100D S131R H168R S174D H168D S174R 12D
G44D K133C A43D F209C G44R S183C A43R+ S176C N Q105R K147R Q100R
S131D Q105D K147D Q100D S131R H168R S174D H168D S174R 13A G44D
F170C A43D S162C G44R K133C A43R+ I117C N Q105R K147R Q100R S131D
Q105D K147D Q100D S131R H168R S174D H168D S174R 13B G44D F170C A43D
S162C G44R K133C A43R+ F209C N Q105R K147R Q100R S131D Q105D K147D
Q100D S131R H168R S174D H168D S174R 13C G44D F170C A43D S162C G44R
V173C A43R+ Q160C N Q105R K147R Q100R S131D Q105D K147D Q100D S131R
H168R S174D H168D S174R 13D G44D F170C A43D S162C G44R S183C A43R
S176C Y Q105R K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D
H168D S174R 14A.sup.& G44D V173C A43D Q160C G44R K133C A43R
I117C N Q105R K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D
H168D S174R 14B.sup.& G44D+ V173C A43D Q160C G44R K133C A43R
F209C N Q105R K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D
H168D S174R 14C.sup.& G44D V173C A43D Q160C G44R+ F170C A43R
S162C Y Q105R K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D
H168D S174R 14D.sup.& G44D V173C A43D Q160C G44R S183C A43R
S176C Y Q105R K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D
H168D S174R 15A G44D F170C A43D S176C G44R K133C A43R I117C N Q105R
K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D H168D S174R
15B G44D F170C A43D S176C G44R K133C A43R F209C N Q105R K147R Q100R
S131D Q105D K147D Q100D S131R H168R S174D H168D S174R 15C G44D
F170C A43D S176C G44R V173C A43R Q160C Y Q105R K147R Q100R S131D
Q105D K147D Q100D S131R H168R S174D H168D S174R 15D G44D F170C A43D
S176C G44R S183C A43R S176C Y Q105R K147R Q100R S131D Q105D K147D
Q100D S131R H168R S174D H168D S174R 16A G44D K133C A43D I117C G44R
F170C A43R S162C N Q105R K147R Q100R S131D Q105D K147D Q100D S131R
H168R S174D H168D S174R 16B G44D K133C A43D I117C G44R F170C A43R
S176C N Q105R K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D
H168D S174R 16C G44D K133C A43D I117C G44R V173C A43R Q160C N Q105R
K147R Q100R S131D Q105D K147D Q100D S131R H168R S174D H168D S174R
16D G44D K133C A43D I117C G44R S183C A43R S176C N Q105R K147R Q100R
S131D Q105D K147D Q100D S131R H168R S174D H168D S174R *A "Y"
indicates that the host cells (1) produced easily detectable
amounts of full-length antibody when DNAs encoding one or both
cognate HC/LC pairs were present and (2) produced little or no
full-length antibody when only DNAs encoding non-cognate HC/LC
pairs were present. Such rows are in boldface. A "N" indicates that
not all these criteria were not met. .sup.#This column provides the
designation for the particular pair of altered variant antibodies.
.sup.&Data shown in FIG. 8.
[0554] To extend these results to a different antibody pair, pairs
of altered antibodies containing the altered IgG4 anti-PD1 102
antibody described in Example 2 and an altered version of the IgG1
anti-CTLA4 111 antibody described above, which contained the
alterations D399K and K409E (to disfavor the formation of HC/HC
heterodimers), were tested in a similar manner. Using some of the
combinations of alterations explored in the chain drop out
experiments reported in Table 20, DNAs encoding altered pairs of
the anti-PD1 and anti-CTLA4 antibodies were made and tested in
chain drop out transfection experiments as described above. These
results are cataloged in Table 21 below.
TABLE-US-00022 TABLE 21 Alterations tested in chain drop out
experiments Accurate anti-PD1 (IgG4) anti-CTLA4 (IgG1; D399K,
K409E) HC/LC HC1 LC1 HC2 LC2 partner Variant.sup.# VH CH1 VL CL VH
CH1 VL CL selection* 17A G44D F170C V43D S162C G44R F170C A43R
S162C N Q105R K147R G100R S131D Q105D K147D P100D S131R H168R S174D
H168D S174R 17B G44D F170C V43D S162C G44R F170C A43R S176C Y Q105R
K147R G100R S131D Q105D K147D P100D S131R H168R S174D H168D S174R
17C G44D F170C V43D S162C G44R V173C A43R Q160C Y Q105R K147R G100R
S131D Q105D K147D P100D S131R H168R S174D H168D S174R 17D G44D
F170C V43D S162C G44R S183C A43R S176C N Q105R K147R G100R S131D
Q105D K147D P100D S131R H168R S174D H168D S174R 18A.sup.& G44D
V173C V43D S162C G44R F170C A43R S162C N Q105R K147R G100R S131D
Q105D K147D P100D S131R H168R S174D H168D S174R 18B.sup.& G44D
V173C V43D S162C G44R F170C A43R S176C Y Q105R K147R G100R S131D
Q105D K147D P100D S131R H168R S174D H168D S174R 18C.sup.& G44D
V173C V43D S162C G44R V173C A43R Q160C Y Q105R K147R G100R S131D
Q105D K147D P100D S131R H168R S174D H168D S174R 18D.sup.& G44D
V173C V43D S162C G44R S183C A43R S176C N Q105R K147R G100R S131D
Q105D K147D P100D S131R H168R S174D H168D S174R 19A G44D S183C V43D
S176C G44R F170C A43R S162C N Q105R K147R G100R S131D Q105D K147D
P100D S131R H168R S174D H168D S174R 19B G44D S183C V43D S176C G44R
F170C A43R S176C N Q105R K147R G100R S131D Q105D K147D P100D S131R
H168R S174D H168D S174R 19C G44D S183C V43D S176C G44R V173C A43R
Q160C Y Q105R K147R G100R S131D Q105D K147D P100D S131R H168R S174D
H168D S174R 19D G44D S183C V43D S176C G44R S183C A43R S176C N Q105R
K147R G100R S131D Q105D K147D P100D S131R H168R S174D H168D S174R
20A G44D K147R V43D S131D G44R K147D A43R S131R N Q105R H168R G100R
S174D Q105D H168D P100D S174R 23A.sup.@ G44D K147R V43D S131D G44R
K147D A43R S131R N Q105R H168R G100R S174D Q105D H168D P100D S174R
23B.sup.@ G44D K147R V43D S131D G44R F170C A43R S176C N Q105R H168R
G100R S174D Q105D K147D P100D S131R H168D S174R 23C.sup.@ G44D
K147R V43D S162C G44R F170C A43R S176C N Q105R H168R G100R S131D
Q105D K147D P100D S131R S174D H168D S174R 23D.sup.@ G44D V173C V43D
S162C G44R F170C A43R S176C Y Q105R K147R G100R S131D Q105D K147D
P100D S131R H168R S174D H168D S174R *A "Y" indicates that the host
cells (1) produced easily detectable amounts of full-length
antibody when DNAs encoding one or both cognate HC/LC pairs were
present and (2) produced little or no full-length antibody when
only DNAs encoding non-cognate HC/LC pairs were present. Such rows
are shown in boldface. A "N" indicates that not all these criteria
were not met. .sup.@Data from these variants are shown in FIG. 9.
.sup.&Data from these variants are shown in FIG. 7. .sup.#This
column provides the designation for the particular pair of altered
variant antibodies.
[0555] These data indicate that some of the same sites for charged
pairs and cysteine pairs that were effective in forcing cognate
HC/LC pairing in the anti-HER2 antibody mixtures containing two
full-length IgG1 antibodies were also effective in the mixtures
described in Table 21 containing both an IgG1 and an IgG4
full-length antibody. Compare 14C and 14D in Table 20 to 17C and
19C in Table 21.
[0556] In addition, the variant mixtures 23A-23D show a clear
effect from the addition of disulfide bonds. The antibodies in the
23A mixture have no added cysteine residues, while the antibodies
in the 23B and 23C mixtures would be expected to have an additional
disulfide bond (due to cysteine substitutions) in the anti-CTLA4
antibody only. The 23D mixture would be expected to have an
additional disulfide bond in each of the two antibodies in the
mixture. Table 21. DNAs encoding these four antibody mixtures
encoded mixtures that were the same except for these differences in
cysteine substitutions. Table 21. Expression of the antibody having
cysteine substitutions in both the HC and LC is strong in the 23B
and 23C mixtures compared to that observed in 23A mixture. FIG. 9,
compare lanes 1 and 4 to lanes 6 and 9 and to lanes 11 and 14.
However, some expression of non-cognate pairs is detectable in
transfectants from mixtures 23B and 23C. FIG. 9, lanes 10 and 15.
In mixture 23D, good expression of full-length antibody was
observed in all transfectants containing DNA encoding cognate HC/LC
pairs (FIG. 9, lanes 16, 17, and 19), and little or no expression
was detected in transfectants containing only DNA encoding
non-cognate pairs (FIG. 9, lanes 18 and 20). These data indicate
that addition of disulfide bonds to antibodies in a mixture
increases both expression (likely due to a stronger HC/LC
interaction that allows escape from the ER) and selectivity of
pairing.
[0557] In further experiments, methods of producing no more than
two or three major species of antibodies in a host cell line
containing DNAs encoding two IgG antibodies, one of which contained
no partner-directing alterations, were explored. In test samples,
one of the antibodies did not contain any partner-directing
alterations, and the other lacked the naturally-occurring HC/LC
disulfide bond (due to the substitutions C220S/G (HC2) and C214S
(LC2)) and, in some samples, had two potential HC/LC disulfide
bonds (due to cysteine substitutions at contacting residues) and,
in some cases, also an HC/LC charge pair (due to substitutions of
charged amino acids at contacting residues). Chain drop out
experiments were performed as explained above using DNAs encoding
the IgG4 anti-PD1 102 antibody (comprising HC1 and LC1) comprising
only the alteration S228P (to prevent Fab arm exchange) and the
IgG1 anti-CTLA 4 antibody 111 (comprising HC2 and LC2), which, in
test samples, was engineered to lack the naturally-occurring
disulfide bridge and comprise alterations disfavoring heterodimers
(D399K and K409E in the HC) plus partner-directing alterations
including alterations potentially creating two HC/LC disulfide
bridges and, in some cases, also an HC/LC charge pair. The cysteine
substitutions tested included the following pairs of alterations in
HC2 and LC2, respectively: H168C and S174C; V173C and 0160C; and
F170C and S162C. The substitutions of charged amino acids tested
included the following pairs of alterations in the HC2 and LC2,
respectively: K147D and S131R/K; and H168D and S174K/R. Samples
were subjected to SDS-PAGE under non-reducing conditions and
further analyzed by Western blot using goat-anti-human IgG Fc
specific polyclonal antibodies for detection as described above.
Results are shown in FIG. 10.
[0558] Cells transfected with DNAs encoding various chains of
unaltered versions of the two antibodies produced full-length IgG
antibodies of about 150 kDa. FIG. 10, panel A, lanes 1-5. These
data indicate that non-cognate HC/LC pairs can produce full-length
antibodies in the absence of alterations preventing such pairing.
Elimination of the naturally-occurring disulfide bond in an
engineered anti-CTLA4 111 antibody (comprising HC2 and LC2)
prevented formation of full-length IgG antibody in any transfectant
that did not contain DNA encoding both chains of the unaltered IgG4
anti-PD1 antibody, i.e., HC1 and LC1. FIG. 10, panel A, lanes 6-10.
Adding two new potential disulfide bonds to an engineered
anti-CTLA4 111 antibody lacking the naturally-occurring disulfide
bond restored the ability of transfected containing DNA encoding
HC2 and LC2 to produce full-length antibody while greatly
decreasing production of full-length IgG antibody when only DNA
encoding non-cognate pairs was present. FIG. 10, panel A, lanes
11-25. Among the combinations of pairs of cysteine substitutions
tested, the alterations F170C plus V173C (HC2) and Q160C plus S162C
(LC2) led to the best expression of full-length IgG in the presence
of a cognate HC2/LC2 pair (compare lane 24 to lanes 14 and 19 in
panel A) and the least expression of full-length IgG in the
presence of only non-cognate HC/LC pairs (compare lanes 23 and 25
to lanes 13 and 15 or lanes 18 and 20 in panel A). However, the
other two combinations of pairs of cysteine substitutions tested
were almost as effective. See lanes 11-20 of FIG. 10, panel A.
These alterations were as follows: H168C plus V173C (HC2) and Q160C
plus S174C (LC2); and H168C plus V170C (HC2) and S162C plus S174C
(LC2). Thus, these results suggest that the elimination of the
naturally-occurring HC/LC disulfide bridge and the addition of two
pairs of cysteine substitutions at contacting residues in the HC
and LC can lead to robust expression of cognate HC/LC pairs and
little expression of non-cognate pairs.
[0559] In another set of experiments, the effect of adding an HC/LC
charge pair, in addition to two disulfide bridges, to the second
antibody (which lacked the naturally-occurring disulfide bridge)
was tested. We chose the conserved (see Tables 7 and 10) contacting
pair of amino acids K147 (HC2) and S131 (LC2) because K147 is a
charged amino acid. We hypothesized that a K147D/E alteration (HC2)
and an S131K/R alteration (LC2) would create pairs of contacting
amino acids having the same polarity in HC1/LC2 pairs and having
unfavorable interactions in HC2/LC1 pairs, thus further
discouraging the formation of non-cognate pairs. As explained in
Example 1, we also hypothesized that changing a charged amino acid
to a different charged amino acid might be tolerated without
causing a steric clash in the HC/LC interface. If such a steric
clash occurred, it might destabilize the antibody. The alterations
would, of course, create an additional charge pair in cognate
HC2/LC2 pairs, thus hopefully, would favor formation of this
cognate pair without destabilizing the antibody.
[0560] Chain drop-out transfections and were performed and analyzed
by Western blotting under non-reducing conditions as described
above. Results are shown in FIG. 10, panel B. As expected,
transfectants containing various combinations of the chains of the
two antibodies, unaltered except for S228P in HC1 and D399K plus
K409E in HC2, produced full-length IgG antibody of about 150 kDa,
regardless of whether cognate or non-cognate HC/LC pairs were
present. FIG. 10, panel B, lanes 1-5. In the presence of the
alterations C220S (HC2) and C214S (LC2), addition of two pairs of
cysteine substitutions to HC2 and LC2 (F170C plus V173C (HC2) and
Q160C plus S162C (LC2)) led to strong expression of full-length IgG
in the presence of only HC2 and LC2 (FIG. 10, panel B, lane 9) and
barely detectable expression of full-length IgG in the presence of
only non-cognate pairs, i.e., HC1/LC2 and HC2/LC1 (FIG. 10, panel
B, lanes 8 and 10). Further addition, of K147D (HC2) and either
S131K or S131R (LC2) led to even less, if any, expression of
full-length IgG in the presence of only non-cognate pairs. FIG. 10,
panel B, lanes 13, 15, 18, and 20. Hence these results, in
combination with those in panel A, suggest that the addition of the
tested charge pairs led to robust expression of the HC2/LC2 pair
and little if any expression of non-cognate pairs.
[0561] In a third set of experiments, the effects of adding a
different charge pair, i.e., H168D (HC2) and S174K/R (LC2), to the
second antibody (lacking the naturally-occurring disulfide bridge
and having two introduced disulfide bridges), optionally along with
K147D (HC2) and S131K/R (LC2), on efficiency and specificity of
HC/LC pairing were tested. As with K147D (HC2) and S131K/R (LC2),
we hypothesized that H168D/E (HC2) and an S174K/R (LC2) would
create pairs of contacting amino acids having the same charge
polarity in HC1/LC2 pairs (since H168 in HC1 and S174K/R in LC2 are
both positively charged) and possibly some repulsion for HC2/LC1
pairing, thus further discouraging the formation of non-cognate
pairings. In addition, these alterations would, of course, create
additional charge pairs in cognate HC2/LC2 pairs, thus potentially
stabilizing this cognate pair.
[0562] Chain drop-out transfections and were performed and analyzed
by Western blotting under non-reducing conditions as described
above. Results are shown in FIG. 10, panel C. When the second
antibody comprises H168D (HC2) and S174K (LC2) (in addition to
lacking the naturally-occurring disulfide bridge and having two
pairs of cysteine substitutions at contacting residues), expression
of cognate HC2/LC2 pairs is robust (FIG. 10, panel C, lane 4),
whereas little if any expression of non-cognate HC/LC pairs is
detected (FIG. 10, panel C, lanes 3 and 5). Similar results are
obtained when the second antibody comprises H168D (HC2) and S174R
(LC2), although expression of cognate HC2/LC2 pairs is not as
robust. FIG. 10, panel C, lanes 6-10. When the second antibody
comprises H168D plus K147D (HC2) and S174K plus S131K (LC2) (in
addition to lacking the naturally-occurring disulfide bridge and
having two pairs of cysteine substitutions at contacting residues),
expression of HC2/LC2 is robust (FIG. 10, panel C, lane 14) and
little or no expression of non-cognate pairs is observed (FIG. 10,
panel C, lanes 13 and 15), an effect that is particularly clear in
lane 13. This last result may be explained by the fact that an
HC1/LC2 pair will, in this situation, contain two pairs of
contacting amino acids having the same charge. Interestingly, when
the second antibody comprises H168D plus K147D (HC2) and S174R plus
S131R (LC2) (in addition to lacking the naturally-occurring
disulfide bridge and having two pairs of cysteine substitutions at
contacting residues), expression of HC2/LC2 pairs is not nearly as
robust (compare FIG. 10, panel C, lane 19 to lane 14), although
expression of non-cognate pairs is similarly very low (FIG. 10,
panel C, lanes 18 and 20).
[0563] Taken together, the results in FIG. 10 suggest the following
conclusions. First, in a host cell transfected with DNAs encoding
two different IgG antibodies, elimination of the
naturally-occurring interchain HC/LC disulfide bridge in the second
of the antibodies essentially prevents expression of that
full-length antibody (HC2/LC2), as well as the expression of
non-cognate pairs including either chain of the second antibody
(HC2/LC1 and HC1/LC2). However, further addition of cysteine
substitutions (potentially creating two new disulfide bridges in
the second antibody) restores expression of HC2/LC2 without
substantially restoring the expression of non-cognate pairs.
Further addition of one or two HC2/LC2 charge pairs to the second
antibody further enhances the expression HC2/LC2 and further
decreases expression of non-cognate pairs. Finally, HC2/LC2 charge
pairs containing lysine plus aspartate led to higher expression of
HC2/LC2 than did charge pairs containing arginine plus aspartate.
Taken together, these data suggest that a host cell transfected
with DNAs encoding two different IgG antibodies can produce
predominantly antibodies having cognate HC/LC pairs, even when only
one of the antibodies comprises partner-directing alterations. In
the absence of alterations that disfavor heterodimers, only three
major species of antibody could potentially be produced, and only
two major species of antibody could potentially be produced in the
presence of alterations that disfavor HC1/HC2 heterodimers.
Example 4: Identifying Alterations that Disfavor HC/HC
Heterodimers
[0564] When DNAs encoding two different full-length antibodies,
i.e., DNAs encoding two different HCs and two different LCs, are
introduced into a host cell, the cell could potentially produce ten
different antibodies having different combinations of HCs and LCs.
FIG. 4; Carter (2001), J. Immunol. Methods 248: 7-15. To reduce the
number of antibody species produced in such a host cell, the two
different HCs could be engineered so that they form mostly, if not
exclusively, HC/HC homodimers, and the HCs and LCs could be
engineered such that mostly or only cognate HC/LC pairings would
occur. Examples 1-3 address this latter issue. This Example
describes making and testing various alterations in HCs designed to
disfavor, if not completely eliminate, HC/HC heterodimer formation
between the two different HCs.
[0565] To identify alterations that would achieve the goal of
eliminating heterodimeric HC/HC pairs, a panel of DNAs encoding
pairs of proteins including one Fc fragment and one cognate HC/LC
pair, which had different alterations in their CH3 domains, were
made. These DNAs were introduced into host cells that could express
them, and the antibodies produced by the host cells were analyzed
by SDS-PAGE and Western blotting as described above to determine
the relative amounts of HC-LC/Fc heterodimers, HC-LC/HC-LC
homodimers, and Fc/Fc homodimers produced by host cells into which
the DNAs were introduced.
[0566] DNA constructs encoding Fc fragments and HC/LC pairs with
different alterations in the CH3 domain were generated using PCR
and Gibson assembly as explained in Examples 2 and 3. The resulting
DNAs encoding the Fc fragment and the full-length HC and LC, which
included different alterations, were co-transfected into HEK293
host cells and expressed using EXPI293.TM. Expression System from
ThermoFisher Scientific (Waltham, Mass., USA). Conditioned media,
which contained the antibodies produced by the host cells, were
harvested from the transfectants after four days of culture.
[0567] The antibodies in the media were detected by Western
blotting under non-reducing conditions as explained in Examples 2
and 3. After visualization using a CHEMIDOC.TM. XRS+ System with
IMAGE LAB.TM. Software from Bio-Rad Laboratories, Inc., IMAGE
LAB.TM. Software was used to calculate the percentage HC-LC/Fc
heterodimers produced by the host cells transfected with DNA
encoding the various pairs of altered proteins.
[0568] FIG. 11 shows Western blots of samples taken from culture
media of transfectants containing pairs of DNAs encoding an LC and
a wild type human IgG4 HC plus an altered human IgG1 Fc fragment
having various single substitutions at position K370 (top right
panel), K392 (top left panel), or V397 (bottom left panel). Each of
these positions is at the CH3/CH3 interface (see, e.g., WO
2015/017548, Table 1 on page 8, which is incorporated herein by
reference), and K370 and K392 are also close to position 409 (which
is an R in IgG4 and a K in IgG1) in the tertiary structure. V397 is
close to K392. Therefore, it was possible that these residues might
play a role in strengthening or weakening interactions between CH3
domains, especially where one heavy chain is an IgG4 and the other
is an IgG1. However, the data in FIG. 11 indicate that none of the
substitutions tested substantially reduced the percentage of
heterodimers (indicated as "HC/Fc") compared to that observed when
the Fc fragment was not altered, even though some of the
substitutions at all three sites replaced the original amino acid
with one that was larger. Compare lane 18 of FIG. 11 to all other
lanes in FIG. 11.
[0569] In further experiments, culture media from transfectants
containing DNAs encoding a human IgG4 HC (which was wild type other
than the single alteration S228P included to prevent Fab arm
exchange) and a human IgG1 Fc fragment with alterations at D399
and/or K409 were analyzed. Results are shown in FIG. 12. Single
substitutions D399K, D399R, K409D, and K409E in the IgG1 Fc
fragment increased heterodimer formation. Compare lane 1 of FIG. 12
to lanes 2-5 of FIG. 12. However, when the IgG1 Fc fragment
contained D399K and K409E or D399K and K409D, heterodimers
comprised less than 5% of the total antibodies produced by the
transfectants. FIG. 12, lanes 6 and 7.
[0570] The data shown in FIG. 13 address the question of whether
use of an IgG4 full-length HC, rather than, for example, an IgG1
HC, played a role in the results shown in FIG. 12. In FIG. 13,
lanes 10-19 show data from transfectants containing DNA encoding a
wild type (lanes 10 and 15) or altered (lanes 11-14 and 16-19)
human IgG1 Fc fragment and two different human IgG4 HCs (one (Ab1)
in lanes 10-14 and the other (Ab2) in lanes 15-19). Data from Ab2
IgG4 (lanes 15-19) and Ab2 IgG1 (lanes 20-24) are directly
comparable since these antibodies contain the same variable domains
in an IgG4 or IgG1 format, respectively. The alterations in the Fc
were at positions D399 and K409, as indicated in the table in FIG.
13. Little or no heterodimer was detected in transfectants
producing an altered Fc fragment and a full-length human IgG4 HC.
FIG. 13, lanes 11-14 and 16-19. In contrast, heterodimers are
detectable in media from transfectants producing an altered Fc
fragment and a full-length human IgG1 HC. FIG. 13, lanes 21-24.
Thus, these data indicate that the use of an IgG4 HC rather than an
IgG1 HC is important for obtaining the lowest levels of
heterodimers.
[0571] As shown in Table 8, a human IgG4 HC has an arginine at
position 409, whereas a human IgG1 HC has a lysine at position 409.
The following experiment investigated whether this difference plays
a role in the effect observed in FIG. 13, i.e., that host cells
producing a human IgG4 HC plus an altered (D399K/R, K409E/D) human
IgG1 Fc made lower levels of HC-LC/Fc heterodimers than did host
cells producing a human IgG1 HC plus the same altered IgG1 Fc. Host
cells were transfected with DNAs encoding a human IgG1 Fc fragment
and a human HC plus LC. The Fc was wild type (WT) or contained
alterations as indicated in FIG. 14. As indicated, the HC was
either a WT IgG1, a WT IgG4, or an IgG1 containing the alteration
K409R, which makes an IgG1 like an IgG4 at this position. The data
in FIG. 14 show that levels of heterodimers (indicated as "HC/Fc")
were lowest when the host cells were producing an Fc containing
D399K/R and K409D/E and an HC that was either a wild type (WT) IgG4
or an IgG1 containing the alteration K409R. Compare lanes 2-5 and
12-15 to lanes 7-10 in FIG. 14. When a WT IgG1 HC was used with an
Fc containing D399K/R and K409D/E, higher levels of heterodimers
were observed. FIG. 14, lanes 7-10. Thus, alterations that
essentially eliminate detectable heterodimer formation have been
identified in these experiments.
Example 5: Assessing Antibody Mixtures by Size
[0572] To determine how many species of antibodies were present in
some of the altered antibody mixtures described above, the sizes of
the antibodies in the mixtures were determined. First, the sizes of
the full-length antibody species in these mixtures were assessed by
SDS-PAGE under non-reducing conditions, and the sizes of individual
HCs and LCs were assessed by SDS-PAGE under reducing conditions.
The presence of multiple bands at about 150 kDa (full-length
antibody) under non-reducing conditions and/or at about 50 kDa (HC)
and/or 25 kDa (LC) under reducing conditions would be supportive of
the concept that mixture contains multiple full-length antibody
species. To further resolve full-length antibody species, cation
exchange (CEX) chromatography was performed at low pH, which
provided clear resolution of species in a size range near 150 kD.
In addition, sizes of Fab fragments from a handful antibody
mixtures were determined by mass spectrometry (MS), which can
distinguish mass differences in the range expected for most of the
different Fab fragments that could possibly form in the antibody
mixtures containing cognate and/or non-cognate HC/LC pairs.
[0573] To assess the sizes of the full-length antibodies and the
individual HCs and LCs produced in cells transfected with DNAs
encoding the variant MabPairs 17B, 17C, 18B, 18C and 19C (see Table
21), EXPI293.TM. cells were transfected with DNAs encoding the HC
and the LC of each of the two antibodies of these MabPairs.
Following transfection, the culture supernatant was harvested after
4 days of incubation with shaking. Antibodies were purified with a
Protein A column, analyzed in 4-15% CRITERION.TM. TGX
STAIN-FREE.TM. Precast SDS-PAGE gel as described in Example 3, and
blotted and detected as described in Example 2. Results are shown
in FIG. 15.
[0574] As indicated, lanes 1-7 of FIG. 15 contain non-reduced
samples, and lanes 8-14 contain reduced samples. As a control,
lanes 1 and 8 contain an antibody mixture expected to contain two
different anti-HER2 antibodies (4D5-8 and 2C4 with alterations
described in Table 20 as variant 14D) plus a bispecific containing
one HC and one LC from each antibody. Under non-reducing
conditions, this sample shows three bands of about 150 kDa,
suggesting the presence of the three different antibodies. As a
further control, lanes 2 and 9 (labeled IgG1) contain an IgG1
anti-CTLA4 antibody, which shows a single band of about 150 kDa
under non-reducing conditions, suggesting that this sample contains
a single antibody species. Under non-reducing conditions, the
MabPair variants 17B, 17C, 18B and 18C showed two bands of about
150 kDa (lanes 3-6), suggesting that these samples contained two
antibody species. This is in line with expectations because one of
the antibodies in these mixtures contains alterations disfavoring
heterodimer formation, and the mixtures would therefore be expected
to be MabPairs, i.e., mixtures containing only two major species of
antibodies. Variant 19C showed only one band under non-reducing
conditions (lane 7), suggesting that multiple antibody species
comigrate in this sample.
[0575] When reduced, the control sample showed one band of about 50
kDa and two bands of about 25 kDa (FIG. 15, lane 8), suggesting
that the two different HCs migrated at same place, whereas the two
different LCs migrated separately. The IgG1 control under reducing
conditions (lane 9) showed a single band of about 50 kDa and a
single band of about 25 kDa. All reduced MabPair variants samples
showed a single band at size of 50 KDa and two bands of about 25
kDa (lanes 10-14), suggesting that the two different HCs migrated
together, whereas the two different LCs migrated separately. Thus,
the data in FIG. 15, when taken together with data in Examples 3
and 4 and the data below, strongly suggest that the 17B, 17C, 18B,
18C, and 19C mixtures contain no more than two major species of
antibodies.
[0576] Since the two IgG1 anti-HER2 antibodies used as a starting
point for designing the variant antibody mixtures 1A-16D (Tables
17-20) did not contain any alterations disfavoring heterodimer
formation, these variant mixtures could contain three different
major antibody species when produced by a host cell, that is, two
different monospecific antibodies containing two identical HCs and
two identical LCs and a bispecific antibody containing two
different HCs and two different LCs. To test this hypothesis, DNAs
encoding both HCs and both LCs of five variant 3-in-1 mixtures,
i.e., 13D, 14C, 14D, 15B, and 15C (see Table 20 and FIG. 8), were
transfected into 30-50 milliliters (ml) of EXPI293.TM. cells in
shaking flasks. The supernatants were harvested and antibodies were
purified using a standard Protein A column. Different amounts of
purified antibodies in 1:2 series dilution starting from 200 ng per
lane were analyzed on 4-15% CRITERION.TM. TGX STAIN-FREE.TM.
Precast SDS-PAGE gel (Bio-Rad Laboratories, Inc.) and blotted and
detected as described in Example 2. Under non-reducing conditions
(FIG. 16, top panel), all purified antibody mixtures showed 3
distinct bands of about 150 kDa, suggesting that these mixtures
contain three different antibodies. Under reducing conditions (FIG.
16, bottom panel), all purified mixtures showed 1 band of around 50
kDa and 2 bands of around 25 kDa, suggesting that the two different
HCs migrate together, whereas the two different LCs migrate
separately.
[0577] To get better resolution of the full-length antibody species
in the size range around 150 kD, low pH cation exchange
chromatography (CEX) was performed on a sample containing the
variant antibody mixture 18C (described in Table 21), which
contains an anti-PD1, anti-CTLA4 MabPair. This method is described
by Chen et al. (2010), Protein Science, 19:1191-1204, which is
incorporated herein in its entirety. Briefly, it employs a Thermo
PROPAC.TM. WCX-10 weak CEX column, 4.times.250 mm, preceded by a 50
mm guard column (PROPAC.TM. WCX-10G) using a Waters Alliance 2695
high performance liquid chromatography (HPLC) system. The
chromatography was run with a linear gradient from 100% Buffer A
(20 mM sodium acetate pH 5.2) to 100% Buffer B (20 mM sodium
acetate with 250 mM sodium chloride pH 5.2) over 30 minutes. The
column is washed with high salt (1M sodium chloride) and
re-equilibrated to starting condition of Buffer A. The sample
contained 18.8 mg of protein, and the antibodies were detected in
the column outflow by absorbance at 214 nm.
[0578] Results are shown in FIG. 17. As indicated in FIG. 17, the
top two tracings result from chromatography of the anti-CTLA4 (top)
and anti-PD1 (second from top) antibodies that were the starting
point for the alterations described in Examples 3 and 4. The bottom
tracing results from chromatography of the variant antibody mixture
18C (see Table 21). The relative percentages of the components in
the 18C mixture were calculated from the areas of the peaks using
EMPOWER.TM. software from Waters Corporation (Milford, Mass., USA),
which also served to control the HPLC system. In the bottom tracing
resulting from the mixture 18C, the earlier, smaller peak
corresponding to the anti-PD1 antibody comprised 21% of the
mixture, and the later, larger peak corresponding to the anti-CTLA4
antibody comprised 79% of the mixture. These data show that low pH
CEX easily distinguishes between different full-length antibodies
species and can be used to quantitate relative amounts of specific
antibody species in a mixture.
[0579] To obtain data that could distinguish cognate from
non-cognate HC/LC pairs, supernatants of transfectants containing
DNA encoding five different variant antibody mixtures (13D, 14C,
14D, 15C, and 15D) were analyzed by MS. The mixtures were digested
with papain (Thermo Scientific, cat no. 44985) to generate Fab
fragments. When IgG molecules are incubated with papain in the
presence of cysteine, one or more peptide bonds in the hinge region
(generally between His224-Thr 225) are broken, producing three
fragments of similar size (about 50 kD): two Fab fragments and one
Fc fragment. Immobilized enzyme is advantageous because digestion
can be immediately stopped with simple separation of the IgG
solution from the papain-coated resin by centrifugation, resulting
in a digest that is essentially enzyme-free. The digestion products
were analyzed on a SYNAPT.TM. G2 MS system (Waters Corporation,
Milford, Mass., USA). Given the high resolution of MS, the Fab and
Fc fragments were easily distinguishable. The analysis described
below focuses on masses near the expected masses of the Fab
fragments.
[0580] Only four different Fab fragments can be made from two
different HCs (HC1 and HC2) and two different LCs (LC1 and LC2),
that is, an HC1/LC1 Fab, an HC1/LC2 Fab, an HC2/LC2 Fab, and an
HC2/LC1 Fab. In most cases, these different Fab fragments can be
separated using MS. Table 22 below shows calculated masses of the
four possible Fab fragments for 13D, 14C, 14D, 15B, and 15C
antibody mixtures.
TABLE-US-00023 TABLE 22 Calculated masses of Fab fragments Variant
HC/LC Calculated mass of mixture combination Fab (daltons)* 13D
HC1/LC1 47868.08 HC2/LC2 47943.22 HC1/LC2 47777.9 HC2/LC1 48044.4
14C HC1/LC1 47875.07 HC2/LC2 47883.12 HC1/LC2 47676.65 HC2/LC1
48081.44 14D HC1/LC1 47875.07 HC2/LC2 47943.22 HC1/LC2 47736.85
HC2/LC1 48081.44 15C HC1/LC1 47868.08 HC2/LC2 47890.12 HC1/LC2
47765.85 HC2/LC1 47992.35 15D HC1/LC1 47868.08 HC2/LC2 47943.22
HC1/LC2 47777.9 HC2/LC1 48033.4 *These mass estimates do not take
into account any potential post-translational modifications such
as, for example, glycosylation. However, no N-glycoscylation is
predicted in these Fab fragments.
[0581] If only cognate HC/LC pairs were present in the 14D mixture,
only two Fab fragments, HC1/LC1 and HC2/LC2 Fabs, would be present
in the Fab fragments recovered from the papain digestion. FIG. 18
shows the results of the MS analysis of the Fab fragments from the
14D antibody mixture. Two major species were detected, one at about
47,877 daltons and the other at about 47,942 daltons. FIG. 18.
These likely are the HC1/LC1 Fab (predicted size 47,875.07 daltons)
and the HC2/LC2 Fab (predicted size 47,943.22 daltons). If
appreciable quantities non-cognate HC/LC pairs had been present in
the mixture, then two other peaks at around 48,081.44 daltons
and/or 47,736.85 daltons would have been detected. Since such peaks
were not detected and would be easily separable from the peaks
actually detected using MS, these data strongly suggest that most
if not all HC/LC pairs in the 14D mixture are cognate HC/LC
pairs.
[0582] Of the four other samples analyzed by MS, the 13D and 15D
mixtures gave results similar to those shown in FIG. 18, i.e., only
two major peaks were observed at almost the same masses as those
calculated for the Fab fragments from cognate HC/LC pairs. In
mixtures 14C and 15C, only one major peak was observed, which was
almost the same mass as that calculated for the Fab fragment from
one of the cognate HC/LC pairs in each of these mixtures. We
hypothesize that the Fab fragment from the other cognate HC/LC pair
in these samples was digested by the papain used to generate the
Fab fragments since the cognate pairs in question formed
full-length antibody in the chain drop-out experiments described
above.
Example 6: Characterization of IgG1/IgG4 MabPairs where One
Antibody Contains No Partner-Directing Alterations
[0583] Data described above suggest that it is possible for a
single host cell line transfected with DNAs encoding two different
IgG antibodies to produce only two major species of antibodies. In
those experiments, one or both antibodies were engineered to
achieve this result. The following experiments were aimed at
further characterizing MabPairs where one of the antibodies
comprises no partner-directing alterations or alterations
disfavoring heterodimers, and the other antibody comprises one or
more partner-directing alterations, as well as one or more
alterations disfavoring heterodimers. To determine the number of
major species formed in transfectants containing DNAs encoding such
antibodies, the following experiments were performed.
[0584] The human IgG1 anti-CTLA4 111 antibody was used as a
starting place to create an engineered antibody. The amino acid
sequences of the constant domains and the framework regions of the
variable domains of anti-CTLA4 111 antibody are identical to those
of anti-CTLA4 antibody 1E1 reported in SEQ ID NO:38 (HC) and SEQ ID
NO:40 (LC). A DNA was constructed that encoded the HC of anti-CTLA4
111 antibody including the following alterations: K147D, F170C,
V173C, C220G, R255K, D399R, and K409E. Generally, IgG antibodies
comprising the alteration R255K are cleared faster from serum as
compared to antibodies without this alteration, and the alterations
D399R and K409E disfavor HC/HC heterodimer formation. Another DNA
was constructed that encoded the light chain of the anti-CTLA4 111
antibody comprising the following alterations: S131K, Q160C, S162C,
and C214S.
[0585] Plasmid DNAs encoding this engineered anti-CTLA4 111
antibody, an unaltered anti-CTLA4 111 antibody, and an unaltered
version of the humanized IgG4 anti-PD1 102 antibody described in
Example 2 were recovered from cultured bacteria containing them and
were purified using a Qiagen.RTM. Midi-prep kit (Qiagen N.V., the
Netherlands). Mammalian EXPI293.TM. cells were transfected with the
plasmid DNAs encoding each antibody separately or encoding a
mixture of antibodies containing the engineered anti-CTLA4 111
antibody and the anti-PD1 102 antibody using LIPOFECTAMINE.RTM.
2000 (ThermoFisher Scientific, Waltham, Mass., USA) in 125-mL
shaking flasks. Cells were continuously shaken at 150 rpm at
37.degree. C. for 4 days. The supernatant was harvested by spinning
down cells at 1500 rpm for 20 min, and antibodies in the
supernatant were purified using a standard Protein A column. The
purified antibodies were analyzed by MS using an Agilent 6224
accurate-mass time-of-flight (TOF) mass spectrometer equipped with
an electrospray ionization (ESI) source. The results are shown in
FIG. 19.
[0586] MS analysis of deglycosylated antibodies resulting from
EXPI293.TM. cells transfected with DNAs encoding the unaltered
anti-CTLA4 111 antibody indicated that this antibody had a mass of
144,890.12 daltons, which is within 7 parts per million (ppm) from
the predicted mass of 144,889.16 daltons. FIG. 19, panel A. MS
analysis of deglycosylated antibodies resulting from EXPI293.TM.
cells transfected with DNAs encoding the engineered anti-CTLA4 111
antibody showed that this antibody had a mass of 144,780.02
daltons, which is within 7 ppm from the predicted mass of
144,779.00 daltons. FIG. 19, panel B. MS analysis of deglycosylated
antibodies resulting from EXPI293.TM. cells transfected with DNAs
encoding the HC and LC of both the anti-PD1 102 antibody and the
engineered anti-CTLA4 111 antibody yielded two major peaks of about
146,000 daltons. A small amount of a species likely comprising one
HC and one LC (73,214.20 daltons) was also detected. FIG. 19, panel
C. Zooming in on the major peaks revealed one peak of 144,779.69
daltons and another of 146,426.58 daltons. FIG. 19, panel D. These
masses match the predicted masses of the engineered anti-CTLA4 111
antibody (144,779.00 daltons) and the anti-PD1 102 antibody
(146,426.20 daltons) within 4.8 ppm and 2.6 ppm, respectively.
Hence, these data indicated that only two major species of antibody
were produced in host cells containing DNA encoding the anti-PD1
102 antibody and the engineered anti-CTLA4 111 antibody.
[0587] The purified pair of antibodies resulting from transfection
of EXPI293.TM. cells with DNAs encoding the HC and LC of both the
anti-PD1 102 antibody and the engineered anti-CTLA4 111 antibody
was further analyzed to confirm that only cognate HC/LC pairs were
present. The pair of antibodies was digested with IdeS Protease
(Promega, cat no. V7511, which cleaves an IgG antibody at a single
site below the hinge region, yielding F(ab').sub.2 fragments and
fragments comprising the CH2 and CH3 domains) and further treated
with 2-mercaptoethyl amine (2-MEA) in the presence of
ethylenediaminetetraacetic acid (EDTA). The treatment with 2-MEA
and EDTA reduces hinge region disulfide bridges without
substantially affecting HC/LC disulfide bridges. Thus, this
treatment would be expected to yield Fab' and fragments comprising
the CH2 and CH3 domains, possibly accompanied by minor quantities
of LC and Fd fragments. LCs and Fd fragments are designated LC1
(anti-PD1) or LC2 (anti-CTLA4) and Fd1 (anti-PD1) or Fd2
(anti-CTLA4), depending on whether they are derived from the first
or second antibody. See FIG. 20, panel A. Potentially, the Fab'
fragments could contain LC1 and Fd1, LC2 and Fd2, LC2 and Fd1,
and/or LC1 and Fd2. The calculated masses of these Fab' fragments
are shown in the table below.
TABLE-US-00024 TABLE 23 Calculated masses of potential Fab'
fragments of anti-PD1, anti-CTLA4 antibody pair Fd/LC Calculated
mass combination of Fab' (daltons) Fd1/LC1 49,460.32 Fd2/LC2
48,607.56 Fd1/LC2 49,272.08 Fd2/LC1 48,795.82
[0588] Analysis of the digested and 2-MEA plus EDTA-treated pair of
antibodies by MS yielded peaks at 48,605.68 and 49,458.94 daltons,
which matched the calculated Fd2/LC2 mass (within 39 ppm) and
Fd1/LC1 Fab' mass (within 28 ppm), respectively. FIG. 20, panel B.
No other peaks were observed in the size range surrounding the
calculated masses of the Fab' fragments. Thus, these data indicate
that both observed HC/LC pairs were cognate pairs.
[0589] To confirm formation of the two newly introduced disulfide
bonds in the engineered anti-CTLA4 111 antibody, 100 .mu.g of this
antibody was incubated in phosphate buffer at pH 7.2 containing 5
mM N-ethylmaleimide (NEM) (ThermoScientific, cat no. 23030) at room
temperature for 2 hours. NEM can alkylate free thiols, e.g.,
cysteine residues that are not part of a disulfide bridge. After
excess NEM was removed by buffer exchange, lysyl endopeptidase
(Wako Chemicals, cat no. 125-05061, which cleaves proteins on the
carboxyl side of lysine residues) was added at a 1:10
(enzyme:protein) ratio, and the reaction mixture was incubated at
37.degree. C. for 16 hrs. Then half of the mixture was reduced
using 25 mM of Tris-(2-carboxyethyl)phosphine (TCEP,
ThermoScientific, cat no. 20490) at room temperature for 30
min.
[0590] Liquid chromatography-mass spectrometry (LC-MS) analysis of
lysyl endopeptidase digestion products was performed as follows.
Reverse phase (RP) HPLC of reduced and non-reduced samples was
performed on an Agilent Polaris C18-A column (2.0.times.250 mm, 5
.mu.m, Agilent Technologies, Inc.). A sample of 50 .mu.g was
injected onto the column, and the column was initially held at 98%
mobile phase A (0.1% trifluoroacetic acid (TFA) in water) and 2%
mobile phase B (0.1% TFA in 90% acetonitrile (ACN)) for 5 minutes.
Separation was achieved with a 2-22% mobile phase B linear gradient
in 40 minutes followed by a 22-52% mobile phase B linear gradient
in 160 minutes. The column temperature and flow rate were
maintained at 50.degree. C. and 0.2 mL/min, respectively.
[0591] Online MS analysis of the column effluent was performed in
positive ion mode on an Agilent 6224 accurate-mass TOF mass
spectrometer equipped with an ESI source. The drying gas
temperature, drying gas flow and nebulizer were set at 350.degree.
C., 12 L/min and 40 psig, respectively. The capillary, fragmentor,
skimmer1 and Oct RF Vpp were set at 4500V, 250V, 60V and 750V,
respectively. The instrument was calibrated in an m/z range of 100
to 3000 at 4 GHz high resolution. Data from LC/MS were analyzed
using Agilent MASSHUNTER.RTM. Qualitative and BioConfirm
software.
[0592] Whether the substituted cysteine residues F170C (HC)-S162C
(LC) and V173C (HC)-Q160C (LC) were actually forming disulfide
bonds in the altered anti-CTLA4 antibody was determined by
comparison of the column profiles and mass analyses of reduced
versus non-reduced lysyl endopeptidase digests. Under non-reducing
conditions, a peak at a retention time of 103.48 minutes on the
reverse phase column was detected (labeled "3 disulfide-linked
peptide," FIG. 21, panel B), but this peak was not present under
reducing conditions (FIG. 21, panel C). If the predicted disulfide
bonds form, the two peptides shown in FIG. 21, panel A would be
covalently linked by one intrachain and two interchain disulfide
bonds after lysyl endopeptidase digestion. The peptide in the peak
detected at 103.48 minutes under non-reducing conditions had a real
monoisotopic mass of 9441.35 daltons (5.times.1889.07-4=9441.35,
since the peptide was in the +5 charge state), which is only 2 ppm
away from the predicted monoisotopic mass of the linked peptides,
i.e., 9441.37 daltons. FIG. 22, panel A. Under reducing conditions,
two peaks were observed at retention times of 39.65 and 113.05
minutes (labeled Chain A and Chain B, respectively), which were not
present under non-reducing conditions. FIG. 21, panel C. These two
peaks had real monoisotopic masses of 7321.48 daltons
(1831.12.times.4-3=7321.48) and 2126.90 daltons
(1063.95.times.2-1=2126.90) (FIG. 22, panels B and C), which
exactly matched the theoretical monoisotopic masses of chains A and
B (of FIG. 21, panel A), respectively.
[0593] In addition, mass analyses of NEM-treated unaltered
anti-CTLA4 versus the engineered anti-CTLA4 antibody discussed
immediately above showed that both had about the same percentage of
free cysteine residues, i.e., cysteine residues that are not part
of a disulfide bridge. Data not shown. Taken together, these data
indicated that the two newly introduced cysteine pairs at F170C
(HC)-5162C (LC) and V173C (HC)-Q160C (LC) do form disulfide
bonds.
Example 7: Characterization of IgG2/IgG4 MabPairs where One
Antibody Contains No Partner-Directing Alterations
[0594] The following experiment was done to determine whether
MabPairs like those described in Example 6, but containing IgG2 and
IgG4 antibodies rather than IgG1 and IgG4 antibodies, could be
successfully made in a single host cell line.
[0595] DNA gBlocks.RTM. (double-stranded DNAs suitable for assembly
by Gibson reaction, among other uses; Integrated DNA Technologies
(IDT), Coralville, Iowa) encoding the HC and LC of an IgG2 antibody
with human constant regions were synthesized by IDT. These were
assembled and inserted into a mammalian expression vector using
Gibson reactions. The HC had the following substitutions: C131S,
K147D, F170C, V173C, D399R, and K409E. The first four of these were
used to enhance cognate HC/LC pairing and discourage non-cognate
HC/LC pairing, and the last two were alterations disfavoring
HC1/HC2 heterodimer formation. The amino acid sequence of the CH1,
hinge, CH2, and CH3 domains of this engineered HC are provided in
SEQ ID NO:42. The LC had the substitutions S131K, Q160C, S162C and
C214S, which were used to enhance cognate HC/LC pairing and
discourage non-cognate HC/LC pairing. The amino acid sequence of
this engineered CL kappa domain is provided in SEQ ID NO:44.
[0596] This engineered IgG2 antibody, either alone or in
combination with an unaltered IgG4 anti-PD1 102 antibody (described
in Example 2), was expressed by transiently transfecting
EXPI293.TM. cells with the plasmid DNAs described above and
culturing the transfected cells. Expressed antibodies were
recovered from the cell supernatants and purified using a Protein A
column. Mass spectrometry of antibodies purified from cells
transfected with DNAs encoding both the IgG2 and IgG4 antibodies
was carried out after deglycosylation by PNGase F. In Table 24
below the calculated masses of full-length deglycosylated IgG
antibodies lacking the C-terminal lysine of the HC (which is mostly
removed by carboxyl peptidase in mammalian cells) resulting from
all possible cognate and non-cognate HC/LC pairings of the IgG2
(comprising HC2 and LC2) and IgG4 (comprising HC1 and LC1)
antibodies described above are shown.
TABLE-US-00025 TABLE 24 Calculated masses of IgG antibodies Chain
Components* Predicted Mass HC1-LC1 and HC1-LC1 146,424.20 HC2-LC2
and HC2-LC2 143,659.64 HC2-LC1 and HC2-LC1 145,116.62 HC1-LC2 and
HC1-LC2 144,967.22 HC1-LC2 and HC2-LC1 145,041.92 HC1-LC1 and
HC2-LC2 145,041.92 HC1-LC1 and HC1-LC2 145,696.71 HC2-LC1 and
HC2-LC2 144,389.13 HC1-LC1 and HC2-LC1 145,771.41 HC1-LC2 and
HC2-LC2 144,314.43 *HC1 and LC1 are the HC and LC from the IgG4
antibody, and HC2 and LC2 are the HC and LC from the IgG2
antibody.
[0597] Actual mass spectrometry results are shown in FIG. 23, panel
A. Two main peaks were detected. The smaller of the two was at
143,666.77 daltons, which matches the calculated mass of the IgG2
antibody (143,659.64 daltons) with an error of 50 ppm. The larger
of the two peaks was at 146,426.81 daltons, which matches the
predicted mass of the IgG4 antibody (146,424.20 daltons) with an
error of 18 ppm. Each of the two main peaks was accompanied by a
shoulder peak at a slightly larger mass, which is believed to be
due to incomplete removal of the HC C-terminal lysine by carboxyl
peptidase in the mammalian cells that produced the antibodies. See
FIG. 23, panel A. Calculated masses of antibodies resulting from
heterodimeric HC/HC pairings and/or non-cognate HC/LC pairing
ranged from 144,314.43 to 145,771.41 daltons. Table 24. No
substantial peaks were observed in this size range (FIG. 23, panel
A), indicating that only two major species of antibodies were
present.
[0598] To confirm that only cognate HC/LC pairs were present, the
purified antibodies were also treated with IdeS Protease, 2-MEA,
and EDTA to generate Fab' fragments and subsequently analyzed by
mass spectrometry analysis as described in Example 6. Table 25
below shows the calculated masses of Fab' fragments resulting from
the four possible Fd/LC pairings, including cognate and non-cognate
pairs.
TABLE-US-00026 TABLE 25 Calculated masses of Fab' fragments Fd/LC
combination* Calculated mass of Fab' (daltons) Fd1/LC1 49,458.32
Fd2/LC2 48,015.87 Fd1/LC2 48,730.83 Fd2/LC1 48,745.36 *Fd1 and LC1
are from the IgG4 antibody, and Fc2 and LC2 are from the IgG2
antibody.
[0599] Actual results from the mass spectrometry are shown in FIG.
23 panel B. Major peaks were detected at 48,017.74 and 48015.87
daltons, which matched the calculated masses for the Fd2/LC2 (with
as error of 39 ppm) and Fd1/LC1 (with an error of 36 ppm) Fab'
fragments. No other peaks at or near the predicted masses of the
Fab' fragments resulting from non-cognate HC/LC pairings, i.e.,
48,730.83 or 48,745.36 daltons, were detected.
[0600] The mass spectrometry results described above indicate that
only two major species of antibodies, both having homodimeric HC/HC
and cognate HC/LC pairings, were produced in cells transfected with
DNAs encoding the engineered IgG2 antibody and the unaltered IgG4
antibody described above. Thus, these results strongly suggest that
the alterations in the IgG2 antibody were sufficient to create a
situation where only two major species of antibodies were produced
in cells transfected DNAs encoding an engineered IgG2 antibody and
an unaltered IgG4 antibody.
Example 8: ELISA-Based Assay to Assess Binding Activity of an
Anti-PD1 and Anti-CTLA4 MabPair Antibody Mixture Produced in a Host
Cell
[0601] The following assay was performed to assess antigen binding
of an anti-PD1, anti-CTLA4 MabPair mixture produced in a single
host cell. Microtiter plates (96 well) were coated with
biotinylated human PD1 (extracellular domain having a
hexa-histidine (His-6) tag) or biotinylated human CTLA4
(extracellular domain having a glutathione S-transferase (GST) tag)
using 100 .mu.l at 1 .mu.g/mL in phosphate buffered saline (PBS).
The plates were washed three times with 1.times.PBST, and blocked
with 250 .mu.l/well of Block Buffer (3% non-fat milk in
1.times.PBST), with shaking at RT for 1 hr. The plates were washed
three more times, and standard antibody control samples or test
samples containing antibody mixtures in Dilution Buffer (PBST plus
0.1% bovine serum albumin (BSA)) were added at 100 .mu.I/well with
shaking at room temperature (RT) for 2 hours. After 3 washes, 100
.mu.l of HRP-conjugated donkey-anti-human IgG at a dilution of
1:5000 in Dilution Buffer was added at to each well, and plates
were shaken for 2 hours at RT. After three washes, 100 .mu.I of
substrate was added to each well, and the plates were incubated
with shaking for 20 minutes. The reaction was stopped by adding
0.16 M sulfuric acid, and the plates were read in an ENVISION.RTM.
(PerkinElmer, Waltham, Mass., USA) plate reader at 450 nm.
[0602] The concentration of anti-PD1 and anti-CTLA4 antibodies in
the tested MabPair mixtures was deduced from standard curves made
using control anti-PD1 and anti-CTLA4 antibodies, respectively. The
results indicated that MabPair variant mixtures 17B, 17C, 18B, 18C,
19C comprised 18.0%, 11.8%, 27.2%, 23.4%, 27.2% anti-PD1 antibody,
respectively, and 82.0%, 88.2%, 72.8%, 76.6%, 72.8% anti-CTLA4
antibody, respectively. Hence, these data indicated that the host
cells producing these variant mixtures produced more anti-CTLA4
antibody than anti-PD1 antibody.
Example 9: Luciferase Reporter Assay to Assess the Potency of
Anti-PD1 Antibody in Anti-PD1 and Anti-CTLA4 MabPair Antibody
Mixture
[0603] The following experiment tests the ability of an anti-PD1
antibody in a MabPair mixture containing anti-PD1 and anti-CTLA4
antibodies to inhibit the interaction of PDL1 and PD1 where the PD1
is expressed on a cell surface.
[0604] Jurkat cells stably expressing PD1 and luciferase (from
Promega Corporation, Madison, Wis., USA) were seeded into 48 wells
of 96-well plates (to avoid edge effects) at 4.times.10.sup.4
cells/well in a volume of 20 RI. Following one day of incubation,
5.times.10.sup.4 CHO cells stably expressing PDL1 in 20 .mu.l were
added to each well, along with 20 .mu.l of a control antibody or a
MabPair mixture. The PDL1 expressed on the CHO cells can engage
with the PD1 on the Jurkat cells, activating an intracellular
signaling pathway that inhibits expression of luciferase. If an
antibody prevents PD1/PDL1 engagement by binding to PD1, luciferase
expression will increase. A 1:3 dilution series of the antibodies
or mixtures was done so that different wells had different
concentrations of the antibody or the mixture. The plates were
incubated at 37.degree. C. in 5% CO.sub.2 for 6 hours. BIO-GLO.TM.
luciferase reagent (Promega, cat. no. G7941) was added at 40 .mu.l
per well to lyse the cells, and plates were read in ENVISION.RTM.
plate reader (PerkinElmer). The results are shown in FIG. 24 and
Table 26 below. These data indicate that the anti-PD1 and
anti-CTLA4 MabPair antibody mixtures 17B, 17C, 18B, 18C and 19C
blocked the PDL1-PD1 interaction with comparable potency compared
to the unaltered anti-PD1 antibody alone (indicated as
"anti-PD1").
TABLE-US-00027 TABLE 26 Potency of anti-PD1 antibody in MabPair
mixtures Percent Anti-PD1 Antibody IC.sub.50 (nM)* 100.0 anti-PD1
1.31 18.0 17B mixture 2.6 11.8 17C mixture 1.47 27.2 18B mixture
2.09 23.4 18C mixture 1.88 27.2 19C mixture 2.44 *Based on the
concentration of anti-PD1 antibody in each mixture as determined in
Example 8.
Example 10: Luciferase Reporter Assay to Assess the Potency of
Anti-CTLA4 Antibody in an Anti-PD1 and Anti-CTLA4 MabPair Antibody
Mixture
[0605] The following experiment tests the ability of an anti-CTLA4
antibody in a MabPair mixture containing anti-PD1 and anti-CTLA4
antibodies to inhibit the interaction of CD80 and/or CD86 with
CTLA4 where the CTLA4 is expressed on a cell surface.
[0606] Jurkat cells stably expressing CTLA4 and luciferase (from
Promega Corporation, Madison, Wis., USA) were seeded into 48 wells
of 96-well microtiter plates at 5.times.10.sup.4 cells/well in a
volume of 15 .mu.l. Following one day of incubation,
5.times.10.sup.4 Raji cells expressing CD80 and CD86 in 15 .mu.l
were added to each well, along with 15 .mu.l of a control
anti-CTLA4 antibody or a MabPair mixture. The CD80 and CD86
expressed on the Raji cells can engage with the CTLA4 on the Jurkat
cells, activating an intracellular signaling pathway that inhibits
expression of luciferase. If an antibody prevents CTLA4 engagement
with CD80 and/or CD86 by binding to CTLA4, luciferase expression
will increase. A 1:3 dilution series of the antibody or the
mixtures was done so that different wells had different
concentrations of the antibody or a mixture. The plates were
incubated at 37.degree. C. in 5% CO.sub.2 for 16 hours. BIO-GLO.TM.
luciferase reagent (Promega, Cat no. G7941) was added at 40 .mu.l
per well to lyse the cells, and plates were read in an
ENVISION.RTM. plate reader (PerkinElmer). Results are shown in FIG.
25 and Table 27 below.
TABLE-US-00028 TABLE 27 Potency of anti-CTLA4 antibody in MabPair
mixtures. Percent IC.sub.50 anti-CTLA4 Antibody (nM)* 100.0
anti-CTLA4 4.594 82.0 17B mixture 23.18 88.2 17C mixture 16.56 72.8
18B mixture 23.56 76.6 18C mixture 8.001 72.8 19C mixture 10.89
*Based on the concentration of anti-CTLA4 antibody in each mixture
as determined in Example 8.
[0607] These data indicate that the anti-CTLA4 antibody in the
anti-PD1 and anti-CTLA4 MabPair mixtures 17B, 17C, 18B, 18C and 19C
blocked the CTLA4-CD80/86 interaction, albeit with less potency
than the unaltered anti-CTLA4 antibody alone. The 18C mixture was
the most potent among the MabPair antibody mixtures. Thus, although
these data indicate that the anti-CTLA4 antibodies in the variant
mixtures had decreased activity (about 2-6 fold less) compared to
the unaltered anti-CTLA4 antibody alone, the MabPair mixtures
containing altered anti-CTLA4 antibodies still did exhibit
anti-CTLA activity. These results may suggest that the alterations
in these anti-CTLA4 antibodies affected their potency, whereas
little change in the potency of the anti-PD1 antibodies was
observed.
Example 11: Luciferase Assay to Assess the Potency of an Anti-CTLA4
Antibody Comprising Partner-Directing Alterations that is Part of a
MabPair Also Including an Anti-PD1 Antibody Lacking Such
Alterations
[0608] This experiment tested the potency of the engineered IgG1
anti-CTLA4 111 antibody, described in Example 6, which comprised
both alterations disfavoring heterodimers and partner-directing
alterations, in the context of a MabPair including the unaltered
anti-PD1 102 antibody described in Example 6. The assay was
performed essentially as described in Example 10. As controls, the
unaltered anti-CTLA4 111 antibody (not as part of a MabPair) and
another unaltered anti-CTLA4 antibody (anti-CTLA4 110 antibody)
with a very closely related sequence were also tested. The amino
acid sequences of the constant domains of the anti-CTLA4 110
antibody are the same as those of the anti-CTLA4 antibody 1E1,
which are provided in amino acids 119-448 of SEQ ID NO:38 (HC) and
108-214 SEQ ID NO:40 (LC). The amino acid sequences of the
framework regions of the variable domains of the anti-CTLA4
antibody 110 are the same as those of the anti-CTLA4 antibody 1E1
except for a single substitution in a framework region. Results are
shown in FIG. 26 and summarized in Table 28 below.
TABLE-US-00029 TABLE 28 Potency of anti-CTLA4 antibody in the
context of MabPair Percent anti-CTLA4 Antibody IC.sub.50 (nM) 100.0
Unaltered anti-CTLA4 110 antibody 8.42 100.0 Unaltered anti-CTLA4
111 antibody 9.13 100.0 Engineered anti-CTLA4 111 antibody 2.79
35.3 MabPair containing an anti-PD1 3.88* antibody and the
engineered anti- CTLA4 111 antibody *Based on the concentration of
anti-CTLA4 antibody in the MabPair mixture, which was determined as
described in Example 8.
[0609] These data indicate that the engineered anti-CTLA4 111
antibody in the MabPair blocked the CTLA4-CD80/86 interaction with
potency comparable to that of the altered anti-CTLA4 111 antibody
alone and slightly higher than that of the unaltered anti-CTLA4 111
or anti-CTLA4 110 antibodies alone. Thus, the presence of the
anti-PD1 antibody in the MabPair did not substantially affect the
potency of the altered anti-CTLA4 111 antibody in this cell-based
assay.
Example 12: Comparison of 3-In-1 Anti-HER2 Antibody Mixtures Versus
a Combination of Two Anti-HER2 Antibodies in Ability to Kill Human
Breast Cancer Cells
[0610] The following experiments were done to determine the
relative abilities of various 3-in-1 antibody mixtures to kill
cancer cells as compared to a combination of the two antibodies
that were the starting place for the creation of the mixtures,
either singly or in combination.
[0611] Human HER2-expressing breast cancer cell lines BT-474 and
SK-BR-3 were seeded at 1.0.times.10.sup.4 cells/mL in 96-well, flat
bottom plates at 100 .mu.L/well. Cells were incubated at 37.degree.
C. at 5% CO.sub.2 for 4 hours. The anti-HER2 antibodies 4D5-8 and
2C4 served as a starting point for making the variant antibody
mixtures 1A-16D. Example 3 and Tables 17-20. Antibody 4D5-8,
antibody 2C4, a combination of these two antibodies, or one of the
3-in-1 antibody mixtures 14D, 15C, 13D, 14C, or 15D (see Table 20)
was added to each well in a volume of 100 .mu.L. A series of 1:4
dilutions starting from 136 nM was done. As a negative control, two
duplicate wells containing a human IgG1/.kappa. antibody believed
to be irrelevant to growth of the cancer cells were included at the
highest antibody concentration tested (136 nM). After 96 hrs of
incubation, 100 .mu.L of supernatant was removed from each well,
and 100 .mu.L of CELL TITER GLO.RTM. reagent (Promega, cat. no.
G7572) was added. The contents were mixed for 2 minutes on an
orbital shaker to induce cell lysis. The plates were incubated at
room temperature for 10 minutes to stabilize the luminescent
signal. The luminescence signals were recorded at 1 sec per well in
an ENVISION.RTM. plate reader (PerkinElmer, Waltham, Mass., USA).
The intensity of the luminescent signal is proportional to the
quantity of ATP present in the cells, which is a reflection of cell
viability. Results are shown in FIG. 27 and in Table 29 below.
TABLE-US-00030 TABLE 29 IC.sub.50's for inhibiting cancer cell
viability of anti-HER2 antibodies or mixtures thereof Antibody or
IC.sub.50 in BT-474 IC.sub.50 in SK-BR-3 mixture cells (nM) cells
(nM) 14D mixture 0.35 0.12 15C mixture 0.22 0.17 13D mixture 0.50
0.42 14C mixture 0.48 0.42 15D mixture 0.46 0.20 4D5-8 + 0.93 0.47
2C4 mixture 4D5-8 0.67 0.02 2C4 >68.0 3.72
[0612] The IgG1/.kappa.LC control antibody (labeled "IgG1" in FIG.
27) did not affect viability of BT-474 cells. The anti-HER2
antibody 2C4 had a slight negative effect on the viability of
BT-474 cells. The anti-HER2 antibody 4D5-8 had a significant
negative effect on BT-474 cell viability, and the combination of
4D5-8 and 2C4 also had a negative effect. The anti-HER2 3-in-1
antibody mixtures 14D, 15C, 13D, 14C, and 15D all had a more
negative effect on BT-474 cell viability than the 4D5-8+2C4
antibody mixture, suggesting that all 3 antibody components (4D5-8,
2C4, and a bispecific antibody that comprises half of each of these
antibodies) inhibit the BT-474 tumor cell growth more than a
combination of only 4D5-8 and 2C4. The 3-in-1 antibody cocktail
sample 15C was the most potent inhibitor in BT-474 cells with
IC.sub.50 of 0.22 nM.
[0613] In SK-BR-3 cells, IgG1/.kappa.LC control antibody did not
affect cell viability. The antibody 2C4 had a small effect SK-BR-3
cell viability, while 4D5-8 had a much larger effect. The
combination of 4D5-8 and 2C4 had a greater effect on viability than
2C4 and a lesser effect than 4D5-8. The anti-HER2 3-in-1 antibody
mixtures 14D, 15C, 13D, 14C, and 15D all had slightly greater
effects on cell viability than the mixture of 2C4+4D5-8. These data
suggest that the antibody species in the 3-in-1 mixtures work
together to inhibit growth of tumor cells.
[0614] These data suggest that a 3-in-1 mixture containing two
different monospecific anti-HER2 antibodies plus a bispecific
antibody comprising half of each antibody can effectively inhibit
growth of HER2-expressing cancer cells.
Sequence CWU 1
1
461132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideConsensus amino acid sequence for a human VH
domainMOD_RES(1)..(3)Any amino acid ordinarily found in living
organismsMOD_RES(5)..(7)Any amino acid ordinarily found in living
organismsMOD_RES(9)..(13)Any amino acid ordinarily found in living
organismsMOD_RES(15)..(16)Any amino acid ordinarily found in living
organismsMOD_RES(17)..(17)serine or
threonineMOD_RES(18)..(18)valine or leucineMOD_RES(19)..(19)Any
amino acid ordinarily found in living
organismsMOD_RES(20)..(20)valine or leucineMOD_RES(21)..(21)serine
or threonineMOD_RES(23)..(25)Any amino acid ordinarily found in
living organismsMOD_RES(27)..(35)Any amino acid ordinarily found in
living organismsMOD_RES(36)..(37)Any amino acid ordinarily found in
living organisms or absentMOD_RES(39)..(39)Any amino acid
ordinarily found in living organismsMOD_RES(42)..(43)Any amino acid
ordinarily found in living organismsMOD_RES(45)..(45)lysine or
glutamineMOD_RES(48)..(48)Any amino acid ordinarily found in living
organismsMOD_RES(50)..(54)Any amino acid ordinarily found in living
organismsMOD_RES(55)..(57)Any amino acid ordinarily found in living
organisms or absentMOD_RES(58)..(70)Any amino acid ordinarily found
in living organismsMOD_RES(72)..(78)Any amino acid ordinarily found
in living organismsMOD_RES(80)..(84)Any amino acid ordinarily found
in living organismsMOD_RES(86)..(93)Any amino acid ordinarily found
in living organismsMOD_RES(95)..(97)Any amino acid ordinarily found
in living organismsMOD_RES(99)..(99)Any amino acid ordinarily found
in living organismsMOD_RES(101)..(108)Any amino acid ordinarily
found in living organismsMOD_RES(109)..(119)Any amino acid
ordinarily found in living organisms or
absentMOD_RES(120)..(121)Any amino acid ordinarily found in living
organismsMOD_RES(123)..(123)Any amino acid ordinarily found in
living organismsMOD_RES(126)..(127)Any amino acid ordinarily found
in living organismsMOD_RES(129)..(129)Any amino acid ordinarily
found in living organismsMOD_RES(132)..(132)Any amino acid
ordinarily found in living organisms 1Xaa Xaa Xaa Leu Xaa Xaa Xaa
Gly Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa
Trp Xaa Arg Gln Xaa Xaa Gly Xaa Gly Leu Xaa 35 40 45Trp Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa
Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa65 70 75 80Xaa
Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa 85 90
95Xaa Tyr Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
100 105 110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Gln Gly Xaa
Xaa Val 115 120 125Xaa Val Ser Xaa 130298PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideConsensus amino acid sequence for a CH1
domainMOD_RES(1)..(5)Any amino acid ordinarily found in living
organismsMOD_RES(7)..(9)Any amino acid ordinarily found in living
organismsMOD_RES(11)..(15)Any amino acid ordinarily found in living
organismsMOD_RES(16)..(16)arginine or lysineMOD_RES(17)..(26)Any
amino acid ordinarily found in living organismsMOD_RES(29)..(29)Any
amino acid ordinarily found in living organismsMOD_RES(31)..(33)Any
amino acid ordinarily found in living organismsMOD_RES(35)..(40)Any
amino acid ordinarily found in living organismsMOD_RES(42)..(50)Any
amino acid ordinarily found in living organismsMOD_RES(52)..(52)Any
amino acid ordinarily found in living organismsMOD_RES(54)..(55)Any
amino acid ordinarily found in living organismsMOD_RES(57)..(63)Any
amino acid ordinarily found in living organismsMOD_RES(65)..(65)Any
amino acid ordinarily found in living organismsMOD_RES(68)..(82)Any
amino acid ordinarily found in living organismsMOD_RES(84)..(98)Any
amino acid ordinarily found in living organisms 2Xaa Xaa Xaa Xaa
Xaa Pro Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Leu Xaa Lys Xaa Xaa 20 25 30Xaa Pro
Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa
Xaa His Xaa Phe Xaa Xaa Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr 50 55
60Xaa Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65
70 75 80Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 85 90 95Xaa Xaa398PRTHomo sapiens 3Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95Lys Val498PRTHomo sapiens 4Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr
Val598PRTHomo sapiens 5Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val698PRTHomo sapiens 6Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val7232PRTHomo sapiens 7Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala1 5 10 15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120
125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220Ser Leu Ser
Leu Ser Pro Gly Lys225 2308228PRTHomo sapiens 8Glu Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val1 5 10 15Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45His Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Met Glu 50 55 60Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr65 70 75
80Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
Pro 100 105 110Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro Gln 115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val 130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200
205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220Ser Pro Gly Lys2259279PRTHomo sapiens 9Glu Leu Lys Thr
Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys1 5 10 15Pro Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 20 25 30Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 35 40 45Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro 50 55
60Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys65
70 75 80Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val 85 90 95Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr
Val Asp 100 105 110Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr 115 120 125Asn Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp 130 135 140Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu145 150 155 160Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg 165 170 175Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 180 185 190Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 195 200
205Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn
210 215 220Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser225 230 235 240Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Ile Phe Ser 245 250 255Cys Ser Val Met His Glu Ala Leu His
Asn Arg Phe Thr Gln Lys Ser 260 265 270Leu Ser Leu Ser Pro Gly Lys
27510229PRTHomo sapiens 10Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser
Cys Pro Ala Pro Glu Phe1 5 10 15Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120
125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu
Gly Lys22511117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideConsensus amino acid sequence for a
human VL domainMOD_RES(1)..(15)Any amino acid ordinarily found in
living organismsMOD_RES(17)..(22)Any amino acid ordinarily found in
living organismsMOD_RES(24)..(27)Any amino acid ordinarily found in
living organismsMOD_RES(28)..(33)Any amino acid ordinarily found in
living organisms or absentMOD_RES(34)..(40)Any amino acid
ordinarily found in living organismsMOD_RES(42)..(48)Any amino acid
ordinarily found in living organismsMOD_RES(49)..(49)alanine,
serine, or prolineMOD_RES(51)..(63)Any amino acid ordinarily found
in living organismsMOD_RES(64)..(64)isoleucine or
valineMOD_RES(66)..(66)Any amino acid ordinarily found in living
organismsMOD_RES(72)..(78)Any amino acid ordinarily found in living
organismsMOD_RES(80)..(89)Any amino acid ordinarily found in living
organismsMOD_RES(90)..(90)alanine or glycineMOD_RES(91)..(91)Any
amino acid ordinarily found in living
organismsMOD_RES(93)..(93)tyrosine or
phenylalanineMOD_RES(94)..(101)Any amino acid ordinarily found in
living organismsMOD_RES(102)..(102)Any amino acid ordinarily found
in living organisms or absentMOD_RES(103)..(104)Any amino acid
ordinarily found in living organismsMOD_RES(107)..(107)glutamine or
glycineMOD_RES(110)..(117)Any amino acid ordinarily found in living
organisms 11Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Gly1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 35 40 45Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 50 55 60Pro Xaa Arg Phe Ser Gly Ser Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Leu Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Phe Gly Xaa Gly Thr Xaa Xaa Xaa 100 105 110Xaa Xaa Xaa Xaa Xaa
11512107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideConsensus amino acid sequence for a CL kappa
domainMOD_RES(1)..(5)Any amino acid ordinarily found in living
organismsMOD_RES(7)..(11)Any amino acid ordinarily found in living
organismsMOD_RES(14)..(23)Any amino acid ordinarily found in living
organismsMOD_RES(25)..(25)Any amino acid ordinarily found in living
organismsMOD_RES(28)..(33)Any amino acid ordinarily found in
living
organismsMOD_RES(35)..(38)Any amino acid ordinarily found in living
organismsMOD_RES(40)..(40)Any amino acid ordinarily found in living
organismsMOD_RES(42)..(52)Any amino acid ordinarily found in living
organismsMOD_RES(54)..(54)Any amino acid ordinarily found in living
organismsMOD_RES(56)..(56)Any amino acid ordinarily found in living
organismsMOD_RES(58)..(66)Any amino acid ordinarily found in living
organismsMOD_RES(68)..(68)Any amino acid ordinarily found in living
organismsMOD_RES(75)..(86)Any amino acid ordinarily found in living
organismsMOD_RES(88)..(90)Any amino acid ordinarily found in living
organismsMOD_RES(92)..(106)Any amino acid ordinarily found in
living organisms 12Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Pro
Pro Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Val Cys
Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Pro Xaa Xaa Xaa Xaa Val Xaa Trp Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Gln Xaa Ser Xaa Thr
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Ser Xaa Ser Ser Thr Leu
Thr Leu Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys 100 10513107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideConsensus amino acid sequence for a CL lambda
domainMOD_RES(1)..(5)Any amino acid ordinarily found in living
organismsMOD_RES(7)..(11)Any amino acid ordinarily found in living
organismsMOD_RES(14)..(23)Any amino acid ordinarily found in living
organismsMOD_RES(25)..(25)Any amino acid ordinarily found in living
organismsMOD_RES(28)..(33)Any amino acid ordinarily found in living
organismsMOD_RES(35)..(38)Any amino acid ordinarily found in living
organismsMOD_RES(40)..(40)Any amino acid ordinarily found in living
organismsMOD_RES(42)..(52)Any amino acid ordinarily found in living
organismsMOD_RES(54)..(54)Any amino acid ordinarily found in living
organismsMOD_RES(56)..(56)Any amino acid ordinarily found in living
organismsMOD_RES(58)..(66)Any amino acid ordinarily found in living
organismsMOD_RES(67)..(67)alanine or methionineMOD_RES(68)..(68)Any
amino acid ordinarily found in living organismsMOD_RES(75)..(86)Any
amino acid ordinarily found in living organismsMOD_RES(88)..(90)Any
amino acid ordinarily found in living
organismsMOD_RES(92)..(106)Any amino acid ordinarily found in
living organisms 13Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Pro
Pro Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Val Cys
Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Pro Xaa Xaa Xaa Xaa Val Xaa Trp Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Glu Xaa Thr Xaa Pro
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Ser Ser Tyr Leu
Ser Leu Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys 100 10514107PRTHomo sapiens 14Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40
45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10515107PRTHomo sapiens 15Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Glu Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val
Tyr Ala Gly Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100 10516107PRTHomo sapiens 16Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10
15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Arg Lys Val Asp Asn Ala Leu
Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Glu Ser Lys
Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 10517107PRTHomo sapiens 17Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His
Lys Leu Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10518107PRTHomo sapiens
18Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1
5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 10519214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideAmino acid sequence of the light
chain of a humanized antibody 19Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu
Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21020450PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 20Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230
235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345
350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys
45021449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideAmino acid sequence of the heavy chain of a
humanized antibody 21Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Thr Asp Tyr 20 25 30Thr Met Asp Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asp Val Asn Pro Asn Ser Gly
Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60Lys Gly Arg Phe Thr Leu Ser
Val Asp Arg Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Leu
Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys22214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideAmino acid sequence of the light chain of a
humanized antibody 22Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val Ser Ile Gly 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21023446PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideAmino acid sequence of the heavy
chain of an engineered antibody 23Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Glu Ile Asp Pro Phe Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55
60Lys Gly Arg Val Thr Met Thr Ile Asp Lys Ser Thr Asn Thr Val Tyr65
70 75 80Met Glu Leu Ser Ser Leu Gly Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Pro Gly Tyr Thr Tyr Gly Gly Met Asp Phe Trp Gly
Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
44524220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideAmino acid sequence of the light chain of an
engineered antibody 24Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser
Gln Ser Leu Phe Ile Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Gly
Ala Ser Thr Arg Asp Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95Asn His Tyr Tyr
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110Lys Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120
125Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu145 150 155 160Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 180 185 190Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215 2202511PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideAmino
acid sequence of a light chain CDR1 of a humanized antibody 25Arg
Ala Ser Gln Asp Val Asn Thr Ala Val Ala1 5 10267PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideAmino
acid sequence of a light chain CDR2 of a humanized antibody 26Ser
Ala Ser Phe Leu Trp Ser1 5279PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideAmino acid sequence of a light
chain CDR3 of a humanized antibody 27Gln Gln His Tyr Thr Thr Pro
Pro Thr1 5285PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideAmino acid sequence of a heavy chain CDR1
of a humanized antibody 28Asp Thr Tyr Ile His1 52917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideAmino
acid sequence of a heavy chain CDR2 of a humanized antibody 29Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys1 5 10
15Gly3011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideAmino acid sequence of a heavy chain CDR3 of a
humanized antibody 30Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr1 5
103111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideAmino acid sequence of a light chain CDR1 of a
humanized antibody 31Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala1 5
10327PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideAmino acid sequence of a light chain CDR2 of a
humanized antibody 32Ser Ala Ser Tyr Arg Tyr Thr1 5339PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideAmino
acid sequence of a light chain CDR3 of a humanized antibody 33Gln
Gln Tyr Tyr Ile Tyr Pro Tyr Thr1 5345PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideAmino
acid sequence of a heavy chain CDR1 of a humanized antibody 34Asp
Thr Tyr Met Asp1 53517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideAmino acid sequence of a heavy
chain CDR2 of a humanized antibody 35Asp Val Asn Pro Asn Ser Gly
Gly Ser Ile Tyr Asn Gln Arg Phe Lys1 5 10 15Gly3610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideAmino
acid sequence of a heavy chain CDR3 of a humanized antibody 36Asn
Leu Gly Pro Ser Phe Tyr Phe Asp Tyr1 5 10371347DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
polynucleotideNucleotide sequence encoding the heavy chain of an
engineered antibody 37caggtgcagc ttgtcgaaag cggtggtggg gtagttgaac
cagggcgttc tttacgttta 60tcttgtgcag cctccggttt caccttcagt tcatacggga
tgcactgggt tagacaagct 120ccaggaaaag gattagaatg ggtagctgtt
atttggtaca accctagcga gaaggattac 180gccgattcag ctaagggtag
gtttaccatt agtagagaca atagtaaaaa cactctatat 240ctacaaatga
acagcttgcg tgccgaggat actgcagttt actactgcgc gagggctggt
300cttctcggtt atttcgacta ctggggtcag gggacattgg taactgtttc
aagcgctagc 360accaagggcc catccgtctt ccccctggcg ccctcctcca
agagcacctc tgggggcaca 420gcggccctgg gctgcctggt caaggactac
ttccccgaac cggtgacggt gtcgtggaac 480tcaggcgccc tgaccagcgg
cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540tactccctca
gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc
600tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga
gcccaaatct 660tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg
aactcctggg gggaccgtca 720gtcttcctct tccccccaaa acccaaggac
accctcatga tctcccggac ccctgaggtc 780acatgcgtgg tggtggacgt
gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtgg
aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg
900taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg
caaggagtac 960aagtgcaagg tctccaacaa agccctccca gcccccatcg
agaaaaccat ctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtac
accctgcccc catcccggga ggagatgacc 1080aagaaccagg tcagcctgac
ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140gagtgggaga
gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac
1200tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag
gtggcagcag 1260gggaacgtct tctcatgctc cgtgatgcat gaggctctgc
acaaccacta cacgcagaag 1320agcctctccc tgtctccggg taaatga
134738448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideAmino acid sequence of the heavy chain of an
engineered antibody 38Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Glu Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asn Pro Ser
Glu Lys Asp Tyr Ala Asp Ser Ala 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Gly
Leu Leu Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 44539645DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
polynucleotideNucleotide sequence encoding the light chain of an
engineered antibody 39gagattgttc tgacacagag tcctggtaca ttatctttgt
cccctggtga aagggcaact 60ctatcttgta gggcctctca atctattagc agctacttgg
cttggtatca acaaaaacca 120ggtcaagcgc cgagaccatt gatttatggt
gtctcctcta gagcaacagg gataccagac 180agatttagtg gaagcggttc
aggtactgat ttcactctaa cgattagccg tttagaacct 240gaagattttg
cagtgtacta ttgtcaacag tacggcatga gcccctttac ctttggtcct
300ggaactaaag tggatataaa gcgtacggtg gctgcaccat ctgtcttcat
cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg
gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag
cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc
600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga
64540214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideAmino acid sequence ofo the light chain of an
engineered antibody 40Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Ile Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Pro Leu Ile 35 40 45Tyr Gly Val Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr Gly Met Ser Pro Phe 85 90 95Thr Phe Gly Pro
Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21041326PRTHomo sapiens 41Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr Val
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105
110Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp 130 135 140Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly145 150 155 160Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg Val Val Ser
Val Leu Thr Val Val His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ala Pro Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn225 230
235 240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile 245 250 255Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 260 265 270Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320Ser Leu Ser Pro
Gly Lys
32542326PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideAmino acid sequence of the CH1, hinge, CH2,
and CH3 domains of an engineered antibody 42Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Arg1 5 10 15Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Asp Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Cys Pro Ala Cys Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75
80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro 100 105 110Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 130 135 140Val Ser His Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly145 150 155 160Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200
205Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile 245 250 255Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro Met Leu Arg Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Glu 275 280 285Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305 310 315
320Ser Leu Ser Pro Gly Lys 32543107PRTHomo sapiens 43Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40
45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10544107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideAmino acid sequence of the CL kappa domain of
an engineered human antibody 44Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Lys
Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Cys Glu
Cys Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 1054572PRTHomo sapiens
45Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Asp Asp Tyr1
5 10 15Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 20 25 30Gly Val His Thr Cys Pro Ala Cys Leu Gln Ser Ser Gly Leu
Tyr Ser 35 40 45Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 50 55 60Tyr Ile Cys Asn Val Asn His Lys65 704620PRTHomo
sapiens 46Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Cys Glu Cys Val
Thr Glu1 5 10 15Gln Asp Ser Lys 20
* * * * *
References