U.S. patent application number 16/820375 was filed with the patent office on 2021-04-01 for bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors.
The applicant listed for this patent is Xencor, Inc.. Invention is credited to Matthew Bernett, Christine Bonzon, John Desjarlais, Michael Hedvat, Gregory Moore.
Application Number | 20210095030 16/820375 |
Document ID | / |
Family ID | 1000005266178 |
Filed Date | 2021-04-01 |
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United States Patent
Application |
20210095030 |
Kind Code |
A1 |
Bernett; Matthew ; et
al. |
April 1, 2021 |
BISPECIFIC IMMUNOMODULATORY ANTIBODIES THAT BIND COSTIMULATORY AND
CHECKPOINT RECEPTORS
Abstract
The present invention is directed to bispecific, heterodimeric
immunomodulatory antibodies.
Inventors: |
Bernett; Matthew; (Monrovia,
CA) ; Moore; Gregory; (Azusa, CA) ;
Desjarlais; John; (Pasadena, CA) ; Hedvat;
Michael; (Encino, CA) ; Bonzon; Christine;
(Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Xencor, Inc. |
Monrovia |
CA |
US |
|
|
Family ID: |
1000005266178 |
Appl. No.: |
16/820375 |
Filed: |
March 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15691665 |
Aug 30, 2017 |
10793632 |
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16820375 |
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62381239 |
Aug 30, 2016 |
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62456033 |
Feb 7, 2017 |
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62479723 |
Mar 31, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/28 20130101;
C07K 2317/55 20130101; C07K 2317/622 20130101; C07K 16/2818
20130101; C07K 2317/31 20130101; A61K 2039/505 20130101; C07K
16/2896 20130101; C07K 16/2803 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1.-25. (canceled)
26. A method for activating T cells for the treatment of cancer,
the method comprising administering to a subject a bispecific
antibody that binds human ICOS and binds a human checkpoint
receptor, wherein the binding to ICOS results in agonism of ICOS
and said binding to said checkpoint receptor results in inhibition
of said checkpoint receptor.
27. The method according to claim 26, wherein said checkpoint
receptor is selected from the group consisting of PD-1, PD-L1,
CTLA-4, LAG-3, and TIM-3.
28. The method according to claim 26, wherein the bispecific
antibody comprises: a) a first heavy chain comprising: i) a first
variant Fc domain; and ii) a first antigen binding domain that is a
single chain Fv region (scFv), wherein said scFv region comprises a
first variable heavy chain, a variable light chain and an scFv
linker, wherein said scFv linker covalently attaches said first
variable heavy chain and said variable light chain; and b) a second
heavy chain comprising a VH-CH1-hinge-CH2-CH3 monomer, wherein VH
is a second variable heavy chain and CH2-CH3 is a second variant Fc
domain; and c) a light chain; wherein said variable heavy domain
and said variable light domain form a second antigen binding
domain, and wherein one of said first and second antigen binding
domains binds human ICOS and the other binds to said checkpoint
receptor.
29. The method according to claim 27, wherein said bispecific
antibody binds ICOS and PD-1.
30. The method according to claim 27, wherein said bispecific
antibody binds ICOS and PD-L1.
31. The method according to claim 27, wherein said bispecific
antibody binds ICOS and CTLA-4.
32. The method according to claim 27, wherein said bispecific
antibody binds ICOS and LAG-3.
33. The method according to claim 27, wherein said bispecific
antibody binds ICOS and TIM-3.
34. The method according to claim 27, wherein said bispecific
antibody binds ICOS and BTLA.
35. The method according to claim 27, wherein said bispecific
antibody binds ICOS and TIGIT.
36. A heterodimeric antibody comprising: a) a first heavy chain
comprising a first Fc domain, an optional domain linker and a first
antigen binding domain comprising an scFv that binds a first
antigen; b) a second heavy chain comprising a heavy chain
comprising a heavy chain constant domain comprising a second Fc
domain, a hinge domain, a CH1 domain and a variable heavy domain;
and c) a light chain comprising a variable light domain and a light
chain constant domain; wherein said variable heavy domain and said
variable light domain form a second antigen binding domain that
binds a second antigen, wherein one of said first and second
antigen binding domains binds human ICOS resulting in agonism of
said ICOS and the other binds human checkpoint receptor and results
in inhibition of said checkpoint receptor, and wherein said antigen
binding domain that binds ICOS comprises a variable heavy domain
comprising a vhCDR1, vhCDR2, and vhCDR3 and a variable light domain
comprising a vlCDR1, vlCDR2, and vlCDR3 selected from the
following: i) a vhCDR1 having SEQ ID NO: 26364, a vhCDR2 having SEQ
ID NO: 26365, a vhCDR3 having SEQ ID NO: 26366, a vlCDR1 having SEQ
ID NO: 26379, a vlCDR2 having SEQ ID NO: 26380, and a vlCDR3 having
SEQ ID NO: 26381, and ii) a vhCDR1 having SEQ ID NO: 26644, a
vhCDR2 having SEQ ID NO: 26645, a vhCDR3 having SEQ ID NO: 26646, a
vlCDR1 having SEQ ID NO: 26659, a vlCDR2 having SEQ ID NO: 26660,
and a vlCDR3 having SEQ ID NO: 26661.
37. A heterodimeric antibody according to claim 36, wherein said
second variant Fc domain comprises amino acid substitutions
N208D/Q295E/N384D/Q418E/N241D, wherein said first and second
variant Fc domains each comprise amino acid substitutions
E233P/L234V/L235A/G236del/S267K; and wherein said first variant Fc
domain comprises amino acid substitutions S364K/E357Q and second
variant Fc domain comprises amino acid substitutions
L368D/K370S,
38. A heterodimeric antibody according to claim 36, wherein said
first and second variant Fc domains comprises a set of
heterodimerization variants selected from the group consisting of
L368D/K370S:S364K/E357Q; L368D/K370S:S364K; L368E/K370S:S364K;
T411E/K360E/Q362E:D401K; and T366S/L368A/Y407V:T366W.
39. A heterodimeric antibody according to claim 36, wherein said
checkpoint receptor is selected from the group consisting of PD-1,
PD-L1, CTLA-4, LAG-3, TIM-3, BTLA, and TIGIT.
40. A heterodimeric antibody according to claim 36, wherein said
first and second variant Fc domains each comprise M428L/N434S.
41. A heterodimeric antibody according to claim 36, wherein said
antigen binding domain that binds ICOS is formed from a variable
light domain and a variable heavy domain selected from the
following: i) variable light domain having SEQ ID NO:26378 and a
variable heavy domain having SEQ ID NO:26363, and ii) variable
light domain having SEQ ID NO:26658 and a variable heavy domain
having SEQ ID NO:26643.
42. A nucleic acid composition comprising: a) a first nucleic acid
encoding a first heavy chain of a heterodimeric antibody according
to claim 36; b) a second nucleic acid encoding a second heavy chain
of the heterodimeric antibody; and c) a third nucleic acid encoding
a light chain of the heterodimeric antibody.
43. An expression vector composition comprising: a) a first
expression vector comprising said first nucleic acid of claim 42;
b) a second expression vector comprising said second nucleic acid
of claim 42; and c) a third expression vector comprising said third
nucleic acid of claim 42.
44. A host cell comprising the expression vector composition of
claim 43.
45. A method of making a heterodimeric antibody comprising
culturing a host cell of claim 44 under conditions wherein said
heterodimeric antibody is expressed, and recovering said antibody.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/691,665, filed Aug. 30, 2017, which claims
priority to U.S. Provisional Patent Application No. 62/381,239,
filed Aug. 30, 2016, U.S. Provisional Patent Application No.
62/456,033, filed Feb. 7, 2017, U.S. Provisional Patent Application
No. 62/479,723, filed Mar. 31, 2017, the contents of which are
expressly fully incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 16, 2020, is named 067461-5198 SL and is 27,219,028
kilobytes in size.
BACKGROUND OF THE INVENTION
[0003] Tumor-reactive T cells lose their cytotoxic ability over
time due to up-regulation of inhibitory immune checkpoints such as
PD-1 and CTLA-4. Two parallel therapeutic strategies are being
pursued for de-repressing tumor-reactive T cells so that they can
continue to kill tumor cells.
[0004] The first approach is immune immunomodulatory blockade by
treating with antagonistic monoclonal antibodies that bind to
either the immunomodulatory itself (PD-1, CTLA-4, etc.) or its
ligand (PD-L1, PD-L2, CD80, CD86, etc.), thus removing the
inhibitory signals holding back tumor-reactive T cells from tumor
cell killing. Immunomodulatory receptors such as CTLA-1, PD-1
(programmed cell death 1), TIM-3 (T cell immunoglobulin and mucin
domain 3), LAG-3 (lymphocyte-activation gene 3), TIGIT (T cell
immunoreceptor with Ig and ITIM domains), and others, inhibit the
activation, proliferation, and/or effector activities of T cells
and other cell types. Guided by the hypothesis that
immunomodulatory receptors suppress the endogenous T cell response
against tumor cells, preclinical and clinical studies of anti-CTLA4
and anti-PD1 antibodies, including nivolumab, pembrolizumab,
ipilimumab, and tremelimumab, have indeed demonstrated that
immunomodulatory blockade results in impressive anti-tumor
responses, stimulating endogenous T cells to attack tumor cells,
leading to long-term cancer remissions in a fraction of patients
with a variety of malignancies. Unfortunately, only a subset of
patients responds these therapies, with response rates generally
ranging from 10 to 30% and sometimes higher for each monotherapy,
depending on the indication and other factors.
[0005] The second approach for de-repressing tumor-reactive T cells
is T cell costimulation by treating with agonistic antibodies that
bind to costimulatory proteins such as ICOS, thus adding a positive
signal to overcome the negative signaling of the immune
checkpoints.
[0006] Accordingly, the invention is directed to bispecific
antibodies that bind to costimulatory receptors (e.g. ICOS, GITR,
OX40, 4-1BB) as well as checkpoint receptors (e.g. PD-1, PD-L1,
CTLA-4, LAG-3, TIM-3, BTLA and TIGIT.
SUMMARY OF THE INVENTION
[0007] Accordingly, in one aspect, the invention provides
bispecific antibodies that monovalently binds a human costimulatory
receptor and monovalently binds a human checkpoint receptor for use
in activating T cells for the treatment of cancer.
[0008] In some aspects, the costimulatory receptor is selected from
the group consisting of ICOS, GITR, OX40 and 4-1BB.
[0009] In additional aspects, the checkpoint receptor is selected
from the group consisting of PD-1, PD-L1, CTLA-4, LAG-3, TIGIT and
TIM-3.
[0010] In further aspects, the antibody binds an antigen pair
selected from: ICOS and PD-1, ICOS and CTLA-4, ICOS and LAG-3, ICOS
and TIM-3, ICOS and PD-L1, ICOS and BTLA, ICOS and TIGIT, GITR and
TIGIT, GITR and PD-1, GITR and CTLA-4, GITR and LAG-3, GITR and
TIM-3, GITR and PD-L1, GITR and BTLA, OX40 and PD-1, OX40 and
TIGIT, OX40 and CTLA-4, IC OX40 OS and LAG-3, OX40 and TIM-3, OX40
and PD-L1, OX40 and BTLA, 4-1BB and PD-1, 4-1BB and CTLA-4, 4-1BB
and LAG-3, 4-1BB and TIM-3, 4-1BB and PD-L1, TIGIT and 4-1BB and
4-1BB and BTLA.
[0011] In additional aspects, the bispecific antibody has a format
selected from those outlined in FIG. 2A-2N.
[0012] In further aspects, the invention provides heterodimeric
antibodies comprising: a) a first heavy chain comprising a first Fc
domain, an optional domain linker and a first antigen binding
domain comprising an scFv that binds a first antigen; b) a second
heavy chain comprising a heavy chain comprising a heavy chain
constant domain comprising a second Fc domain, a hinge domain, a
CH1 domain and a variable heavy domain; and c) a light chain
comprising a variable light domain and a light chain constant
domain; wherein said variable heavy domain and said variable light
domain form a second antigen binding domain that binds a second
antigen, wherein one of said first and second antigen binding
domains binds human ICOS and the other binds human PD-1.
[0013] In an additional aspect, the invention provides
heterodimeric bispecific antibodies comprising: a) a first heavy
chain comprising: i) a first variant Fc domain; and ii) a single
chain Fv region (scFv) that binds a first antigen, wherein said
scFv region comprises a first variable heavy chain, a variable
light chain and a charged scFv linker, wherein said charged scFv
linker covalently attaches said first variable heavy chain and said
variable light chain; and b) a second heavy chain comprising a
VH-CH1-hinge-CH2-CH3 monomer, wherein VH is a second variable heavy
chain and CH2-CH3 is a second variant Fc domain; and c) a light
chain; wherein said second variant Fc domain comprises amino acid
substitutions N208D/Q295E/N384D/Q418E/N241D, wherein said first and
second variant Fc domains each comprise amino acid substitutions
E233P/L234V/L235A/G236del/S267K; and wherein said first variant Fc
domain comprises amino acid substitutions S364K/E357Q and second
variant Fc domain comprises amino acid substitutions L368D/K370S,
wherein one of said first and second antigen binding domains binds
human ICOS and the other binds human PD-1, and wherein numbering is
according to the EU index as in Kabat.
[0014] In some aspects the heterodimeric antibodies have first and
second variant Fc domains that each comprise M428L/N434S.
[0015] In an additional aspect, the invention provides
heterodimeric antibodies comprising: a) a first heavy chain
comprising: i) a first variant Fc domain; and ii) a single chain Fv
region (scFv) that binds a first antigen, wherein said scFv region
comprises a first variable heavy chain, a variable light chain and
a charged scFv linker, wherein said charged scFv linker covalently
attaches said first variable heavy chain and said variable light
chain; and b) a second heavy chain comprising a
VH-CH1-hinge-CH2-CH3 monomer, wherein VH is a second variable heavy
chain and CH2-CH3 is a second variant Fc domain; and c) a light
chain; wherein said first and second variant Fc domains comprises a
set of heterodimerization variants selected from the group
consisting of L368D/K370S:S364K/E357Q; L368D/K370S:S364K;
L368E/K370S:S364K; T411E/K360E/Q362E:D401K; and
T366S/L368A/Y407V:T366W, and wherein one of said first and second
antigen binding domains binds human ICOS and the other binds human
PD-1, and wherein numbering is according to the EU index as in
Kabat.
[0016] In a further aspect, the invention provides nucleic acid
compositions comprising nucleic acids that encode the heterodimeric
antibodies of the invention, expression vector compositions
comprising the nucleic acids, and host cells comprising the
expression vector compositions.
[0017] In an additional aspect, the invention provides
heterodimeric antibodies for use in the activation of T cells for
the treatment of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 presents expression data (RNAseq V2 RSEM) of PD-1 and
T cell costimulatory receptors for bladder, breast, colon, head
& neck, kidney, lung-adeno, lung squamous, ovarian, pancreatic,
prostate, and melanoma cancer compiled from The Cancer Genome Atlas
(TCGA). The square of the Pearson correlation coefficient was
calculated for PD-1 against T cell costimulatory receptors.
[0019] FIG. 2A to 2N depict several formats for the bispecific
antibodies of the present invention. The first is the "bottle
opener" format, with a first and a second anti-antigen binding
domain. Additionally, mAb-Fv, mAb-scFv, Central-scFv, Central-Fv,
one armed central-scFv, one scFv-mAb, scFv-mAb dual scFv format are
all shown. FIG. 2J depicts the "central-scFv2" format, with two
Fab-scFv arms, wherein the Fabs bind a first antigen and the scFvs
bind a second antigen. FIG. 2K depicts the bispecific mAb format,
with a first Fab arm binding a first antigen and a second Fab arm
binding a second antigen. FIG. 2L depicts the DVD-IgG format (see,
e.g., U.S. Pat. No. 7,612,181, hereby expressly incorporated by
reference and as discussed below). FIG. 2M depicts the Trident
format (see, e.g., WO 2015/184203, hereby expressly incorporated by
reference and as discussed below). For all of the scFv domains
depicted, they can be either N- to C-terminus variable
heavy-(optional linker)-variable light, or the opposite. In
addition, for the one armed scFv-mAb, the scFv can be attached
either to the N-terminus of a heavy chain monomer or to the
N-terminus of the light chain.
[0020] FIG. 3A-3B depicts the sequences of XENP23104, a bottle
opener format with the ICOS as the Fab side ([ICOS]_H0.66_L0) and
the PD-1 as the scFv (1G6_L1.94_H1.279), and includes the
M428L/434S variant to extend serum half life. The CDRs are
underlined, the scFv linker is double underlined (in the sequences,
the scFv linker is a positively charged scFv (GKPGS).sub.4 linker,
although as will be appreciated by those in the art, this linker
can be replaced by other linkers, including uncharged or negatively
charged linkers, some of which are depicted in FIG. 8), and the
slashes indicate the border(s) of the variable domains. In
addition, the naming convention illustrates the orientation of the
scFv from N- to C-terminus; some of the sequences herein are
oriented as V.sub.H-scFv linker-V.sub.L (from N- to C-terminus),
while some are oriented as V.sub.L-scFv linker-V.sub.H (from N- to
C-terminus), although as will be appreciated by those in the art,
these sequences may also be used in the opposition orientation from
their depiction herein. As noted herein and is true for every
sequence herein containing CDRs, the exact identification of the
CDR locations may be slightly different depending on the numbering
used as is shown in Table 1, and thus included herein are not only
the CDRs that are underlined but also CDRs included within the
V.sub.H and V.sub.L domains using other numbering systems.
[0021] FIG. 4A-4F depict useful pairs of heterodimerization variant
sets (including skew and pI variants). On FIGS. 4C and F, there are
variants for which there are no corresponding "monomer 2" variants;
these are pI variants which can be used alone on either monomer, or
included on the Fab side of a bottle opener, for example, and an
appropriate charged scFv linker can be used on the second monomer
that utilizes a scFv as the second antigen binding domain. Suitable
charged linkers are shown in FIG. 8.
[0022] FIG. 5 depict a list of isosteric variant antibody constant
regions and their respective substitutions. pI_(-) indicates lower
pI variants, while pI_(+) indicates higher pI variants. These can
be optionally and independently combined with other
heterodimerization variants of the invention (and other variant
types as well, as outlined herein).
[0023] FIG. 6 depict useful ablation variants that ablate
Fc.gamma.R binding (sometimes referred to as "knock outs" or "KO"
variants). Generally, ablation variants are found on both monomers,
although in some cases they may be on only one monomer.
[0024] FIG. 7A-7B show two particularly useful embodiments of the
invention. The "non-Fv" components of this embodiment is shown in
FIG. 9A, although the other formats of FIG. 9 can be used as
well.
[0025] FIG. 8A-8B depict a number of charged scFv linkers that find
use in increasing or decreasing the pI of heterodimeric antibodies
that utilize one or more scFv as a component. The (+H) positive
linker finds particular use herein. A single prior art scFv linker
with single charge is references as "Whitlow", from Whitlow et al.,
Protein Engineering 6(8):989-995 (1993). It should be noted that
this linker was used for reducing aggregation and enhancing
proteolytic stability in scFvs.
[0026] FIG. 9A-9D show the sequences of several useful bottle
opener format backbones based on human IgG1, without the Fv
sequences (e.g. the scFv and the vh and vl for the Fab side).
Bottle opener backbone 1 is based on human IgG1 (356E/358M
allotype), and includes the S364K/E357Q:L368D/K370S skew variants,
the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and
the E233P/L234V/L235A/G236del/S267K ablation variants on both
chains. Bottle opener backbone 2 is based on human IgG1 (356E/358M
allotype), and includes different skew variants, the
N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and the
E233P/L234V/L235A/G236del/S267K ablation variants on both chains.
Bottle opener backbone 3 is based on human IgG1 (356E/358M
allotype), and includes different skew variants, the
N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and the
E233P/L234V/L235A/G236del/S267K ablation variants on both chains.
Bottle opener backbone 4 is based on human IgG1 (356E/358M
allotype), and includes different skew variants, the
N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and the
E233P/L234V/L235A/G236del/S267K ablation variants on both chains.
Bottle opener backbone 5 is based on human IgG1 (356D/358L
allotype), and includes the S364K/E357Q:L368D/K370S skew variants,
the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and
the E233P/L234V/L235A/G236del/S267K ablation variants on both
chains. Bottle opener backbone 6 is based on human IgG1 (356E/358M
allotype), and includes the S364K/E357Q:L368D/K370S skew variants,
N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and the
E233P/L234V/L235A/G236del/S267K ablation variants on both chains,
as well as an N297A variant on both chains. Bottle opener backbone
7 is identical to 6 except the mutation is N297S. Alternative
formats for bottle opener backbones 6 and 7 can exclude the
ablation variants E233P/L234V/L235A/G236del/S267K in both chains.
Backbone 8 is based on human IgG4, and includes the
S364K/E357Q:L368D/K370S skew variants, the
N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and the
E233P/L234V/L235A/G236del/S267K ablation variants on both chains,
as well as a S228P (EU numbering, this is S241P in Kabat) variant
on both chains that ablates Fab arm exchange as is known in the
art. Alternative formats for bottle opener backbone 8 can exclude
the ablation variants E233P/L234V/L235A/G236del/S267K in both
chains Backbone 9 is based on human IgG2, and includes the
S364K/E357Q:L368D/K370S skew variants, the
N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side. Backbone
10 is based on human IgG2, and includes the S364K/E357Q:L368D/K370S
skew variants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the
Fab side as well as a S267K variant on both chains.
[0027] As will be appreciated by those in the art and outlined
below, these sequences can be used with any vh and vl pairs
outlined herein, with one monomer including a scFv (optionally
including a charged scFv linker) and the other monomer including
the Fab sequences (e.g. a vh attached to the "Fab side heavy chain"
and a vl attached to the "constant light chain"). That is, any Fv
sequences outlined herein for anti-CTLA-4, anti-PD-1, anti-LAG-3,
anti-TIM-3, anti-TIGIT and anti-BTLA, whether as scFv (again,
optionally with charged scFv linkers) or as Fabs, can be
incorporated into these FIG. 37 backbones in any combination. The
constant light chain depicted in FIG. 9A can be used for all of the
constructs in the figure, although the kappa constant light chain
can also be substituted.
[0028] It should be noted that these bottle opener backbones find
use in the Central-scFv format of FIG. 1F, where an additional,
second Fab (vh-CH1 and vl-constant light) with the same antigen
binding as the first Fab is added to the N-terminus of the scFv on
the "bottle opener side".
[0029] Included within each of these backbones are sequences that
are 90, 95, 98 and 99% identical (as defined herein) to the recited
sequences, and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
additional amino acid substitutions (as compared to the "parent" of
the Figure, which, as will be appreciated by those in the art,
already contain a number of amino acid modifications as compared to
the parental human IgG1 (or IgG2 or IgG4, depending on the
backbone). That is, the recited backbones may contain additional
amino acid modifications (generally amino acid substitutions) in
addition to the skew, pI and ablation variants contained within the
backbones of this figure.
[0030] FIG. 10A-10E depicts the sequences for a select number of
anti-PD-1 antibodies. It is important to note that these sequences
were generated based on human IgG1, with an ablation variant
(E233P/L234V/L235A/G236del/S267K, "IgG1_PVA_/S267K") which is
depicted in FIG. 6A. The CDRs are underlined. As noted herein and
is true for every sequence herein containing CDRs, the exact
identification of the CDR locations may be slightly different
depending on the numbering used as is shown in Table 1, and thus
included herein are not only the CDRs that are underlined but also
CDRs included within the VH and VL domains using other numbering
systems.
[0031] FIG. 11A-11E depict a select number of PD-1 ABDs, with
additional anti-PD-1 ABDs being listed as SEQ 1-2392, 3125-3144,
4697-7594 and 4697-21810. The CDRs are underlined, the scFv linker
is double underlines (in the sequences, the scFv linker is a
positively charged scFv (GKPGS).sub.4 linker although as will be
appreciated by those in the art, this linker can be replaced by
other linkers, including uncharged or negatively charged linkers,
some of which are depicted in FIG. 8), and the slashes indicate the
border(s) of the variable domains. In addition, the naming
convention illustrates the orientation of the scFv from N- to
C-terminus; some of the sequences in this Figure are oriented as
VH-scFv linker-VL (from N- to C-terminus), while some are oriented
as VL-scFv linker-VH (from N- to C-terminus), although as will be
appreciated by those in the art, these sequences may also be used
in the opposition orientation from their depiction herein. As noted
herein and is true for every sequence herein containing CDRs, the
exact identification of the CDR locations may be slightly different
depending on the numbering used as is shown in Table 1, and thus
included herein are not only the CDRs that are underlined but also
CDRs included within the VH and VL domains using other numbering
systems. Furthermore, as for all the sequences in the Figures,
these VH and VL sequences can be used either in a scFv format or in
a Fab format.
[0032] FIG. 12A-12PP depict a select number of CTLA-4 ABDs, with
additional anti-CTLA-4 ABDs being listed as SEQ ID NO:2393-2414 and
3737-3816. The CDRs are underlined, the scFv linker is double
underlines (in the sequences, the scFv linker is a positively
charged scFv (GKPGS).sub.4 linker although as will be appreciated
by those in the art, this linker can be replaced by other linkers,
including uncharged or negatively charged linkers, some of which
are depicted in FIG. 8), and the slashes indicate the border(s) of
the variable domains. In addition, the naming convention
illustrates the orientation of the scFv from N- to C-terminus; some
of the sequences in this Figure are oriented as VH-scFv linker-VL
(from N- to C-terminus), while some are oriented as VL-scFv
linker-VH (from N- to C-terminus), although as will be appreciated
by those in the art, these sequences may also be used in the
opposition orientation from their depiction herein. As noted herein
and is true for every sequence herein containing CDRs, the exact
identification of the CDR locations may be slightly different
depending on the numbering used as is shown in Table 1, and thus
included herein are not only the CDRs that are underlined but also
CDRs included within the VH and VL domains using other numbering
systems. Furthermore, as for all the sequences in the Figures,
these VH and VL sequences can be used either in a scFv format or in
a Fab format.
[0033] FIG. 13A-13N depict a select number of LAG-3 ABDs, with
additional anti-LAG-3 ABDs being listed as SEQ ID NO:2415-2604 and
3817-3960. The CDRs are underlined, the scFv linker is double
underlines (in the sequences, the scFv linker is a positively
charged scFv (GKPGS).sub.4 linker (SEQ ID NO: XXX), although as
will be appreciated by those in the art, this linker can be
replaced by other linkers, including uncharged or negatively
charged linkers, some of which are depicted in FIG. 8), and the
slashes indicate the border(s) of the variable domains> In
addition, the naming convention illustrates the orientation of the
scFv from N- to C-terminus; some of the sequences in this Figure
are oriented as VH-scFv linker-VL (from N- to C-terminus), while
some are oriented as VL-scFv linker-VH (from N- to C-terminus),
although as will be appreciated by those in the art, these
sequences may also be used in the opposition orientation from their
depiction herein. As noted herein and is true for every sequence
herein containing CDRs, the exact identification of the CDR
locations may be slightly different depending on the numbering used
as is shown in Table 1, and thus included herein are not only the
CDRs that are underlined but also CDRs included within the VH and
VL domains using other numbering systems. Furthermore, as for all
the sequences in the Figures, these VH and VL sequences can be used
either in a scFv format or in a Fab format.
[0034] FIG. 14A-14I depicts the sequences for a select number of
anti-TIM-3 antibodies. It is important to note that these sequences
were generated based on human IgG1 backbone, with an ablation
variant (E233P/L234V/L235A/G236del/S267K, "IgG1_PVA_/S267K")
although other formats can be used as well. The CDRs are
underlined. As noted herein and is true for every sequence herein
containing CDRs, the exact identification of the CDR locations may
be slightly different depending on the numbering used as is shown
in Table 1, and thus included herein are not only the CDRs that are
underlined but also CDRs included within the V.sub.H and V.sub.L
domains using other numbering systems.
[0035] FIG. 15A-15C depicts the sequences for a select number of
anti-PD-L1 antibodies. It is important to note that these sequences
were generated based on human IgG1 backbone, with an ablation
variant (E233P/L234V/L235A/G236del/S267K, "IgG1 PVA/S267K") as
outlined herein, although other formats can be used as well. The
CDRs are underlined. As noted herein and is true for every sequence
herein containing CDRs, the exact identification of the CDR
locations may be slightly different depending on the numbering used
as is shown in Table 1, and thus included herein are not only the
CDRs that are underlined but also CDRs included within the VH and
VL domains using other numbering systems.
[0036] FIG. 16 depicts the sequences for a prototype anti-4-1 BB
antibody. It is important to note that these sequences were
generated based on human IgG1 backbone, with an ablation variant
(E233P/L234V/L235A/G236del/S267K, "IgG1_PVA_/S267K"), although the
other formats can be used as well. The CDRs are underlined. As
noted herein and is true for every sequence herein containing CDRs,
the exact identification of the CDR locations may be slightly
different depending on the numbering used as is shown in Table 1,
and thus included herein are not only the CDRs that are underlined
but also CDRs included within the VH and VL domains using other
numbering systems.
[0037] FIG. 17 depicts the sequences for a prototype anti-OX40
antibody. It is important to note that these sequences were
generated based on human IgG1 backbone, with an ablation variant
(E233P/L234V/L235A/G236del/S267K, "IgG1_PVA_/S267K"), although
other formats can be used as well. The CDRs are underlined. As
noted herein and is true for every sequence herein containing CDRs,
the exact identification of the CDR locations may be slightly
different depending on the numbering used as is shown in Table 1,
and thus included herein are not only the CDRs that are underlined
but also CDRs included within the VH and VL domains using other
numbering systems.
[0038] FIG. 18 depicts the sequences for a prototype anti-GITR
antibody. It is important to note that these sequences were
generated based on human IgG1 backbone, with an ablation variant
(E233P/L234V/L235A/G236del/S267K, "IgG1_PVA_/S267K"), although
other formats can be used as well. The CDRs are underlined. As
noted herein and is true for every sequence herein containing CDRs,
the exact identification of the CDR locations may be slightly
different depending on the numbering used as is shown in Table 1,
and thus included herein are not only the CDRs that are underlined
but also CDRs included within the VH and VL domains using other
numbering systems.
[0039] FIG. 19A-19G depicts the sequences for prototype anti-ICOS
antibodies. It is important to note that these sequences were
generated based on human IgG1 backbone, with an ablation variant
(E233P/L234V/L235A/G236del/S267K, "IgG1_PVA_/S267K"), although
other formats can be used as well. The CDRs are underlined. As
noted herein and is true for every sequence herein containing CDRs,
the exact identification of the CDR locations may be slightly
different depending on the numbering used as is shown in Table X,
and thus included herein are not only the CDRs that are underlined
but also CDRs included within the VH and VL domains using other
numbering systems.
[0040] FIGS. 20A-20G depicts sequences for exemplary anti-ICOS
Fabs. The CDRs are underlined and slashes (/) indicate the
border(s) of the variable regions. As noted herein and is true for
every sequence herein containing CDRs, the exact identification of
the CDR locations may be slightly different depending on the
numbering used as is shown in Table X, and thus included herein are
not only the CDRs that are underlined but also CDRs included within
the VH and VL domains using other numbering systems. Furthermore,
as for all the sequences in the Figures, these VH and VL sequences
can be used either in a scFv format or in a Fab format. It is
important to note that these sequences were generated using
six-histidine (His6 or HHHHHH)(SEQ ID NO: 28666) C-terminal tags at
the C-terminus of the heavy chains, which have been removed.
[0041] FIG. 21A-21B depicts melting temperatures (T.sub.m) and
changes in melting temperature from the parental Fab (XENP22050) as
determined by DSF of variant anti-ICOS Fabs engineered for
stability.
[0042] FIG. 22A-22C depicts equilibrium dissociation constants
(KD), association rates (ka), and dissociation rates (kd) of
variant anti-ICOS Fabs for murine Fc fusions of human ICOS captured
on AMC biosensors as determined by Octet.
[0043] FIG. 23 depicts equilibrium dissociation constants (KD),
association rates (ka), and dissociation rates (kd) of variant
anti-ICOS Fabs for biotinylated IgG1 Fc fusions of human ICOS
captured on SA biosensors as determined by Octet.
[0044] FIG. 24A-24M depicts sequences for exemplary anti-ICOS
scFvs. The CDRs are underlined, the scFv linker is double underline
(in the sequences, the scFv linker is a positively charged scFv
(GKPGS).sub.4 linker (SEQ ID NO: 28849), although as will be
appreciated by those in the art, this linker can be replaced by
other linkers, including uncharged or negatively charged linkers,
some of which are depicted in Figure X), and slashes (/) indicate
the border(s) of the variable regions. The naming convention
illustrates the orientation of the scFv from N- to C-terminus; some
of the sequences in this Figure are oriented as VH-scFv linker-VL
(from N- to C-terminus, see FIG. 24), while some are oriented as
VL-scFv linker-VH (from N- to C-terminus, see FIG. 24B), although
as will be appreciated by those in the art, these sequences may
also be used in the opposition orientation from their depiction
herein. As noted herein and is true for every sequence herein
containing CDRs, the exact identification of the CDR locations may
be slightly different depending on the numbering used as is shown
in Table X, and thus included herein are not only the CDRs that are
underlined but also CDRs included within the VH and VL domains
using other numbering systems. Furthermore, as for all the
sequences in the Figures, these VH and VL sequences can be used
either in a scFv format or in a Fab format. It is important to note
that these sequences were generated using polyhistidine (His6 or
HHHHHH) (SEQ ID NO: 28666) C-terminal tags at the C-terminus of the
heavy chains, which have been removed.
[0045] FIG. 25 depicts melting temperatures (T.sub.m) and changes
in melting temperature from the parental scFv (XENP24352; oriented
as VH-scFv linker-VL from N- to C-terminus) as determined by DSF of
variant anti-ICOS scFvs engineered for stability.
[0046] FIG. 26A-26D depicts the amino acid sequences of prototype
anti-costim.times.anti-checkpoint antibodies in the bottle-opener
format (Fab-scFv-Fc). The antibodies are named using the Fab
variable region first and the scFv variable region second,
separated by a dash, followed by the chain designation (Fab-Fc
heavy chain, scFv-Fc heavy chain or light chain). CDRs are
underlined and slashes indicate the border(s) of the variable
regions. The scFv domain has different orientations (N- to
C-terminus) of either V.sub.H-scFv linker-V.sub.L or V.sub.L-scFv
linker-V.sub.H as indicated, although this can be reversed. In
addition, each sequence outlined herein can include or exclude the
M428L/N434S variants in one or preferably both Fc domains, which
results in longer half-life in serum.
[0047] FIG. 27 depicts induction of cytokine secretion by prototype
costim/checkpoint bottle-openers in an SEB-stimulated PBMC
assay.
[0048] FIG. 28 depicts induction of IL-2 secretion in naive
(non-SEB stimulated) and SEB-stimulated PBMCs following treatment
with the indicated test articles.
[0049] FIG. 29 depicts a schematic associated with the benefit of a
costim.times.checkpoint blockade bispecific antibody, showing that
because tumor TILs co-express immune checkpoint receptors and
costimulatory receptors, a bispecific antibody increases
specificity, enhancing anti-tumor activity and avoiding peripheral
toxicity.
[0050] FIG. 30 depicts that double-positive cells are selectively
occupied by exemplary anti-ICOS.times.anti-PD-1 antibody
(XENP20896) as compared to one-arm anti-PD-1 antibody (XENP20111)
and one-arm anti-ICOS antibody (XENP20266).
[0051] FIG. 31A-31B shows the receptor occupancy of
anti-ICOS.times.anti-PD-1 bispecific antibody (XENP20896), one-arm
anti-ICOS antibody (XENP20266) and one-arm anti-PD-1 antibody
(XENP20111) on A) PD-1 and ICOS double-positive T cells and B) PD-1
and ICOS double-negative T cells after SEB stimulation of human
PBMCs.
[0052] FIG. 32A-32D depicts the amino acid sequences of exemplary
anti-ICOS.times.anti-PD-1 antibodies in the bottle-opener format
(Fab-scFv-Fc). The antibodies are named using the Fab variable
region first and the scFv variable region second, separated by a
dash, followed by the chain designation (Fab-Fc heavy chain,
scFv-Fc heavy chain or light chain). CDRs are underlined and
slashes indicate the border(s) of the variable regions. The scFv
domain has different orientations (N- to C-terminus) of either
V.sub.H-scFv linker-V.sub.L or V.sub.L-scFv linker-V.sub.H as
indicated, although this can be reversed. In addition, each
sequence outlined herein can include or exclude the M428L/N434S
variants in one or preferably both Fc domains, which results in
longer half-life in serum.
[0053] FIG. 33A-33C depicts the amino acid sequences of exemplary
anti-ICOS.times.anti-PD-1 antibodies in the bottle-opener format
(Fab-scFv-Fc) which include the M428L/N434S variants in one or
preferably both Fc domains, which results in longer half-life in
serum. The antibodies are named using the Fab variable region first
and the scFv variable region second, separated by a dash, followed
by the chain designation (Fab-Fc heavy chain, scFv-Fc heavy chain
or light chain). CDRs are underlined and slashes indicate the
border(s) of the variable regions. The scFv domain has different
orientations (N- to C-terminus) of either V.sub.H-scFv
linker-V.sub.L or V.sub.L-scFv linker-V.sub.H as indicated,
although this can be reversed.
[0054] FIG. 34A-34C depicts equilibrium dissociation constants
(KD), association rates (ka), and dissociation rates (kd) of
variant anti-ICOS.times.anti-PD-1 bispecific antibodies for murine
Fc fusion of human ICOS captured on AMC biosensors as determined by
Octet.
[0055] FIG. 35 depicts equilibrium dissociation constants (KD),
association rates (ka), and dissociation rates (kd) of variant
anti-ICOS.times.anti-PD-1 bispecific antibodies for biotinylated
IgG1 Fc fusions of human and ICOS captured on SA/SAX biosensors as
determined by Octet.
[0056] FIG. 36A-36B depicts cell surface binding of variant
anti-ICOS.times.anti-PD-1 bispecific to human T cells in
SEB-stimulated PBMC assays in two separate experiments depicted in
A) and B).
[0057] FIG. 37 shows the receptor occupancy of variant
anti-ICOS.times.anti-PD-1 bispecific antibodies, one-arm anti-ICOS
antibodies and one-arm anti-PD-1 antibody (XENP20111) on PD-1 and
ICOS double-positive T cells after SEB stimulation of human
PBMCs.
[0058] FIG. 38A-38B show that variant anti-ICOS.times.anti-PD-1
bispecific antibodies promote A) IL-2 and B) IFN.gamma. secretion
from SEB stimulated PBMCs.
[0059] FIG. 39A-39B show that variant anti-ICOS.times.anti-PD-1
bispecific antibodies promote A) IL-2 and B) IFN.gamma. secretion
from SEB-stimulated PBMCs.
[0060] FIG. 40A-40B depicts the concentration of IFN.gamma. in mice
on Day A) 7 and B) 11 after engraftment with human PBMCs and
treatment with the indicated test articles.
[0061] FIG. 41A-41B depicts CD45+ cell counts in mice as determined
by flow cytometry on Day A) 11 and B) 14 after engraftment with
human PBMCs and treatment with the indicated test articles.
[0062] FIG. 42A-42B depicts A) CD8+ T cell and B) CD4+ T cell
counts in mice as determined by flow cytometry on Day 14 after
engraftment with human PBMCs and treatment with the indicated test
articles (**p.ltoreq.0.01).
[0063] FIG. 43 depicts the change in body weight in mice by Day 14
after engraftment with human PBMCs and treatment with the indicated
test articles (**p.ltoreq.0.01).
[0064] FIG. 44A-44B depicts the concentration of IFN.gamma. in mice
on Day A) 7 and B) 14 after engraftment with human PBMCs and
treatment with the indicated test articles.
[0065] FIG. 45 depicts CD45+ cell counts in mice as determined by
flow cytometry on Day 14 after engraftment with human PBMCs and
treatment with the indicated test articles.
[0066] FIG. 46A-46C depicts A) CD8+ T cell and B) CD4+ T cell
counts in mice as determined by flow cytometry on Day 14 after
engraftment with human PBMCs and treatment with the indicated test
articles."
[0067] FIG. 47A-47B depicts the change in body weight in mice by
Day 12 and 15 after engraftment with human PBMCs and treatment with
the indicated test articles.
[0068] FIG. 48A-48D depicts the amino acid sequences of exemplary
anti-ICOS.times.anti-PD-1 antibodies in the bottle-opener format
(Fab-scFv-Fc) with alternative ICOS ABDs. The antibodies are named
using the Fab variable region first and the scFv variable region
second, separated by a dash, followed by the chain designation
(Fab-Fc heavy chain, scFv-Fc heavy chain or light chain). CDRs are
underlined and slashes indicate the border(s) of the variable
regions. The scFv domain has different orientations (N- to
C-terminus) of either VH-scFv linker-VL or VL-scFv linker-VH as
indicated, although this can be reversed. In addition, each
sequence outlined herein can include or exclude the M428L/N434S
variants in one or preferably both Fc domains, which results in
longer half-life in serum.
[0069] FIG. 49 depict cytokine release assay for IL-2 after
SEB-stimulation of human PBMCs and treatment with alternative
anti-ICOS.times.anti-PD-1 bispecific antibodies.
[0070] FIG. 50 depict cytokine release assay for IL-2 (as fold
induction over bivalent anti-RSV mAb) after SEB-stimulation of
human PBMCs and treatment with alternative
anti-ICOS.times.anti-PD-1 bispecific antibodies.
[0071] FIG. 51 depicts AKT phosphorylation in SEB-stimulated
purified CD3+ T cells after treatment with
anti-ICOS.times.anti-PD-1 bispecific antibodies.
[0072] FIG. 52 depicts fold induction of A) IL-17A, B) IL17F, C)
IL-22, D) IL-10, E) IL-9, and F) IFN.gamma. gene expression by the
indicated test articles over induction by bivalent anti-RSV as
determined by NanoString.
[0073] FIG. 53A-53F depict mean fold induction in expression of
selected immune response genes by indicated test articles over
treatment with bivalent anti-RSV mAb as determined by NanoString.
The shading intensity corresponds to the magnitude of the fold
change.
[0074] FIG. 54 depicts CD45+ cell counts in mice as determined by
flow cytometry on Day 14 after engraftment with human PBMCs and
treatment with the indicated test articles.
[0075] FIG. 55A-55D, similar to FIG. 9 and FIG. 75, depicts the
sequences of the "backbone" portion (e.g. without the Fvs) of a
number of additional formats, including the Central scFv of FIG.
2F, the Central-scFv2 format of FIG. 2J, the bispecific mAb of FIG.
2K, the DVD-Ig of FIG. 2L and the Trident format of FIG. 2M. In
FIG. 2L, the DVD-Ig.RTM. linkers are shown with double underlining,
with other linkers found in WO2007/024715, hereby incorporated by
reference in its entirety and in particular for those sequences. In
the Trident format, other Trident linkers and coil-coil sequences
are shown in WO 2015/184203, hereby incorporated by reference in
its entirety and in particular for those sequences. As will be
appreciated by those in the art, bolded domains (e.g. "VH1",
VH2-scFv linker-VL2'', etc.) are separated with slashes "/", and
may include optional domain linkers as needed. All of these
backbones utilize the kappa constant region for the light chain,
although the lambda chain can also be used. As for FIG. 9 and FIG.
75, these backbones can be combined with any vh and vl domains as
outlined herein.
[0076] FIG. 56A-56B depicts the amino acid sequence of illustrative
anti-PD-1.times.anti-ICOS antibodies in the bottle-opener format
(Fab-scFv-Fc). The antibodies are named using the Fab variable
region first and the scFv variable region second, separated by a
dash, followed by the chain designation (Fab-Fc heavy chain,
scFv-Fc heavy chain or light chain). CDRs are underlined and
slashes indicate the border(s) of the variable regions. The scFv
domain has different orientations (N- to C-terminus) of either
VH-scFv linker-VL or VL-scFv linker-VH as indicated, although this
can be reversed. In addition, each sequence outlined herein can
include or exclude the M428L/N434S variants in one or preferably
both Fc domains, which results in longer half-life in serum.
[0077] FIG. 57A-57C depicts the amino acid sequence of illustrative
anti-PD-1.times.anti-ICOS antibodies in the central-scFv format.
The antibodies are named using the first Fab-Fc variable region
first and the Fab-scFv-Fc variable region second, separated by a
dash, followed by the chain designation (Fab-Fc heavy chain,
Fab-scFv-Fc heavy chain or light chain). CDRs are underlined and
slashes indicate the border(s) of the variable regions. The scFv
domain has different orientations (N- to C-terminus) of either
VH-scFv linker-VL or V.sub.L-scFv linker-V.sub.H as indicated,
although this can be reversed. In addition, each sequence outlined
herein can include or exclude the M428L/N434S variants in one or
preferably both Fc domains, which results in longer half-life in
serum.
[0078] FIG. 58 depicts the amino acid sequence of illustrative
anti-PD-1.times.anti-ICOS antibodies in the central-scFv2 format.
The antibodies are named using the Fab variable region first and
the scFv variable region second, followed by the chain designation
(heavy chain or light chain). CDRs are underlined and slashes
indicate the border(s) of the variable regions. The scFv domain has
different orientations (N- to C-terminus) of either VH-scFv
linker-VL or VL-scFv linker-VH as indicated, although this can be
reversed. In addition, each sequence outlined herein can include or
exclude the M428L/N434S variants in one or preferably both Fc
domains, which results in longer half-life in serum.
[0079] FIG. 59 depicts the amino acid sequence of an illustrative
anti-PD-1.times.anti-ICOS antibody in the bispecific mAb format.
The antibodies are named using the first Fab variable region for a
first antigen and the second Fab variable region for a second
antigen, separated by a dash, followed by the chain designation
(Heavy Chain 1 or Light Chain 1 for the first antigen and Heavy
Chain 2 or Light Chain 2 for the second antigen). CDRs are
underlined and slashes indicate the border(s) of the variable
regions. Each sequence outlined herein can include or exclude the
M428L/N434S variants in one or preferably both Fc domains, which
results in longer half-life in serum.
[0080] FIG. 60 depicts the amino acid sequence of an illustrative
anti-PD-1.times.anti-ICOS antibody in the DVD-IgG format. The
antibodies are named using the first variable region for a first
antigen and the second Fab variable region for a second antigen,
followed by the chain designation (Heavy Chain or Light Chain).
CDRs are underlined and slashes indicate the border(s) of the
variable regions. Each sequence outlined herein can include or
exclude the M428L/N434S variants in one or preferably both Fc
domains, which results in longer half-life in serum.
[0081] FIG. 61A-61B depicts the amino acid sequence of an
illustrative anti-PD-1.times.anti-ICOS antibody in the Trident
format. The antibodies are named using the VL and VH of a first
antigen which comprises a DART and the Fab variable region for a
second antigen, separated by a dash, followed by the chain
designation (Heavy Chain or Light Chain). CDRs are underlined and
slashes indicate the border(s) of the variable regions. Each
sequence outlined herein can include or exclude the M428L/N434S
variants in one or preferably both Fc domains, which results in
longer half-life in serum.
[0082] FIG. 62 depicts induction of cytokine secretion (IL-2) by
alternative format costim.times.checkpoint blockade bispecific
antibodies in an SEB-stimulated PBMC assay.
[0083] FIG. 63 depicts the amino acid sequences of an illustrative
anti-ICOS.times.anti-CTLA-4 antibody in the bottle-opener format
(Fab-scFv-Fc). The antibodies are named using the Fab variable
region first and the scFv variable region second, separated by a
dash, followed by the chain designation (Fab-Fc heavy chain,
scFv-Fc heavy chain or light chain). CDRs are underlined and
slashes indicate the border(s) of the variable regions. The scFv
domain has different orientations (N- to C-terminus) of either
VH-scFv linker-VL or VL-scFv linker-VH as indicated, although this
can be reversed. In addition, each sequence outlined herein can
include or exclude the M428L/N434S variants in one or preferably
both Fc domains, which results in longer half-life in serum.
[0084] FIG. 64A-64B depicts the amino acid sequence of illustrative
anti-LAG-3.times.anti-ICOS antibodies in the bispecific mAb format.
The antibodies are named using the first Fab variable region for a
first antigen and the second Fab variable region for a second
antigen, separated by a dash, followed by the chain designation
(Heavy Chain 1 or Light Chain 1 for the first antigen and Heavy
Chain 2 or Light Chain 2 for the second antigen). CDRs are
underlined and slashes indicate the border(s) of the variable
regions. Each sequence outlined herein can include or exclude the
M428L/N434S variants in one or preferably both Fc domains, which
results in longer half-life in serum.
[0085] FIG. 65 depicts the amino acid sequence of an illustrative
anti-TIM-3.times.anti-ICOS antibody in the bispecific mAb format.
The antibodies are named using the first Fab variable region for a
first antigen and the second Fab variable region for a second
antigen, separated by a dash, followed by the chain designation
(Heavy Chain 1 or Light Chain 1 for the first antigen and Heavy
Chain 2 or Light Chain 2 for the second antigen). CDRs are
underlined and slashes indicate the border(s) of the variable
regions. Each sequence outlined herein can include or exclude the
M428L/N434S variants in one or preferably both Fc domains, which
results in longer half-life in serum.
[0086] FIG. 66A-66C depicts the amino acid sequences of
anti-ICOS.times.anti-PD-L1 antibodies in the bottle-opener format
(Fab-scFv-Fc) and central-scFv2 format. The bottle-openers are
named using the Fab variable region first and the scFv variable
region second, separated by a dash, followed by the chain
designation (Fab-Fc heavy chain, scFv-Fc heavy chain or light
chain). Central-scFv2s are named using the Fab variable region
first and the scFv variable region second, followed by the chain
designation (heavy chain or light chain). CDRs are underlined and
slashes indicate the border(s) of the variable regions. CDRs are
underlined and slashes indicate the border(s) of the variable
regions. The scFv domain has different orientations (N- to
C-terminus) of either VH-scFv linker-VL or VL-scFv linker-VH as
indicated, although this can be reversed. In addition, each
sequence outlined herein can include or exclude the M428L/N434S
variants in one or preferably both Fc domains, which results in
longer half-life in serum.
[0087] FIG. 67 depicts induction of cytokine secretion (IL-2) by
additional costim.times.checkpoint blockade bispecific antibodies
in an SEB-stimulated PBMC assay.
[0088] FIGS. 68A-68G depict amino acid sequences for exemplary
one-arm anti-ICOS Fab-Fc antibodies. CDRs are underlined and
slashes indicate the border(s) of variable regions. These are
referred to as "one-arm" or "one armed" formats as one amino acid
chain is only an Fc domain, with the other side being an anti-ICOS
Fab side. The Fc domain contains the S364K/E357Q skew variants, as
well as the pI(-)_Isosteric_A variants depicted in Figure X. The
Fab Fc domain contains the L368D/K370S skew variants as well as the
pI ISO(+RR) variants depicted in Figure X. Both Fc domains include
the ablation variants (E233P/L234V/L235A/G236del/S267K).
[0089] FIG. 69 depicts equilibrium dissociation constants (KD),
association rates (ka), and dissociation rates (kd) of variant
one-arm anti-ICOS Fab-Fc antibodies for murine Fc fusions of human
ICOS captured on AMC biosensors as determined by Octet.
[0090] FIG. 70 depicts AKT phosphorylation in SEB-stimulated
purified CD3+ T cells after treatment with bivalent and monovalent
anti-PD-1 antibodies and anti-ICOS.times.anti-PD-1 bispecific
antibodies.
[0091] FIG. 71 depicts AKT phosphorylation in purified CD3+ T cells
after treatment with monovalent anti-ICOS Fab-Fc antibodies with
alternative anti-ICOS ABDs.
[0092] FIG. 72A-72C depict some prototype bispecific antibodies
(OX40.times.PD-1, GITR.times.PD-1, 4-1BB.times.PD-1,
CTLA-4.times.ICOS).
[0093] FIG. 73A-73H depict some prototype mAbs (4-1BB, OX40, GITR,
ICOS, PD-L1 and PD-1), the Fvs of which can be used in combination
with the other Fvs of the invention and in any format (bottle
opener, mAb-Fv, mAb-scFv, central-scFv, bispecific mAb, central-Fv,
one armed central-scFv, one armed scFv-mAb, dual scFv, DVD-Ig or
Trident). Some additional ICOS.times.PD-L1 bottle opener sequences
are shown as well.
[0094] FIG. 74A-74F depict additional PD-1.times.ICOS bottle
openers, in some cases with the PD-1 Fv being in the Fab format and
the ICOS Fv in a scFv format and in other cases reversed.
[0095] FIG. 75A-75D shows the sequences of a mAb-scFv backbone of
use in the invention, to which the Fv sequences of the invention
are added. mAb-scFv backbone 1 is based on human IgG1 (356E/358M
allotype), and includes the S364K/E357Q:L368D/K370S skew variants,
the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and
the E233P/L234V/L235A/G236del/S267K ablation variants on both
chains. Backbone 2 is based on human IgG1 (356D/358L allotype), and
includes the S364K/E357Q:L368D/K370S skew variants, the
N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and the
E233P/L234V/L235A/G236del/S267K ablation variants on both chains.
Backbone 3 is based on human IgG1 (356E/358M allotype), and
includes the S364K/E357Q:L368D/K370S skew variants,
N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and the
E233P/L234V/L235A/G236del/S267K ablation variants on both chains,
as well as an N297A variant on both chains. Backbone 4 is identical
to 3 except the mutation is N297S. Alternative formats for mAb-scFv
backbones 3 and 4 can exclude the ablation variants
E233P/L234V/L235A/G236del/S267K in both chains. Backbone 5 is based
on human IgG4, and includes the S364K/E357Q:L368D/K370S skew
variants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab
side and the E233P/L234V/L235A/G236del/S267K ablation variants on
both chains, as well as a S228P (EU numbering, this is S241P in
Kabat) variant on both chains that ablates Fab arm exchange as is
known in the art Backbone 6 is based on human IgG2, and includes
the S364K/E357Q:L368D/K370S skew variants, the
N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side. Backbone
7 is based on human IgG2, and includes the S364K/E357Q:L368D/K370S
skew variants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the
Fab side as well as a S267K variant on both chains.
[0096] As will be appreciated by those in the art and outlined
below, these sequences can be used with any vh and vl pairs
outlined herein, with one monomer including both a Fab and an scFv
(optionally including a charged scFv linker) and the other monomer
including the Fab sequence (e.g. a vh attached to the "Fab side
heavy chain" and a vl attached to the "constant light chain"). That
is, any Fab sequences outlined herein for anti-CTLA-4, anti-PD-1,
anti-LAG-3, anti-TIM-3, anti-TIGIT, anti-BTLA, anti-ICOS,
anti-GITR, anti-OX40 and anti-4-1BB, whether as scFv (again,
optionally with charged scFv linkers) or as Fabs, can be
incorporated into this FIG. 75A-75D backbone in any combination.
The monomer 1 side is the Fab-scFv pI negative side, and includes
the heterodimerization variants L368D/K370S, the isosteric pI
variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, (all relative to IgG1). The
monomer 2 side is the scFv pI positive side, and includes the
heterodimerization variants 364K/E357Q. However, other skew variant
pairs can be substituted, particularly [S364K/E357Q:L368D/K370S];
[L368D/K370S:S364K]; [L368E/K370S:S364K];
[T411T/E360E/Q362E:D401K]; [L368D/K370S:S364K/E357L],
[K370S:S364K/E357Q], [T366S/L368A/Y407V:T366W] and
[T366S/L368A/Y407V/Y394C:T366W/S354C].
[0097] The constant light chain depicted in FIG. 75A can be used
for all of the constructs in the figure, although the kappa
constant light chain can also be substituted.
[0098] It should be noted that these mAb-scFv backbones find use in
the both the mAb-Fv format of FIG. 1H (where one monomer comprises
a vl at the C-terminus and the other a vh at the C-terminus) as
well as the scFv-mAb format (with a scFv domain added to the
C-terminus of one of the monomers).
[0099] Included within each of these backbones are sequences that
are 90, 95, 98 and 99% identical (as defined herein) to the recited
sequences, and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
additional amino acid substitutions (as compared to the "parent" of
the Figure, which, as will be appreciated by those in the art,
already contain a number of amino acid modifications as compared to
the parental human IgG1 (or IgG2 or IgG4, depending on the
backbone). That is, the recited backbones may contain additional
amino acid modifications (generally amino acid substitutions) in
addition to the skew, pI and ablation variants contained within the
backbones of this figure.
[0100] FIG. 76A-76F depict a number of prior art sequences for Fvs
that bind human PD-1 as vh and vl sequences. As will be appreciated
by those in the art, any of these Fvs can be combined with an Fv
that binds a costimulatory receptor (e.g. ICOS, GITR, OX40 or
4-1BB, including the Fv sequences contained herein) and in any
format (bottle opener, mAb-Fv, mAb-scFv, central-scFv, bispecific
mAb, central-Fv, one armed central-scFv, one armed scFv-mAb, dual
scFv, DVD-Ig or Trident). In particular they can be combined with
ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.
[0101] FIGS. 77A-77B depict a number of prior art sequences for Fvs
that bind human ICOS as vh and vl sequences. As will be appreciated
by those in the art, any of these Fvs can be combined with an Fv
that binds a checkpoint receptor (e.g. PD-1, PD-L1, CTLA-4, TIM-3,
LAG-3, TIGIT and BTLA, including the Fv sequences contained herein)
and in any format (bottle opener, mAb-Fv, mAb-scFv, central-scFv,
bispecific mAb, central-Fv, one armed central-scFv, one armed
scFv-mAb, dual scFv, DVD-Ig or Trident). In particular they can be
combined with PD-1 ABDs having the identifiers 1G6_H1.279_L1.194;
1G6_H1.280_L1.224; 1G6_L1.194_H1.279; 1G6_L1.210_H1.288; and
2E9_H1L1.
[0102] FIG. 78A-78J depict a number of prior art sequences for Fvs
that bind human PD-L1 as vh and vl sequences. As will be
appreciated by those in the art, any of these Fvs can be combined
with an Fv that binds a costimulatory receptor (e.g. ICOS, GITR,
OX40 or 4-1BB, including the Fv sequences contained herein) and in
any format (bottle opener, mAb-Fv, mAb-scFv, central-scFv,
bispecific mAb, central-Fv, one armed central-scFv, one armed
scFv-mAb, dual scFv, DVD-Ig or Trident). In particular they can be
combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.
[0103] FIG. 79A-79B depict a number of prior art sequences for Fvs
that bind human CTLA-4 as vh and vl sequences. As will be
appreciated by those in the art, any of these Fvs can be combined
with an Fv that binds a costimulatory receptor (e.g. ICOS, GITR,
OX40 or 4-1BB, including the Fv sequences contained herein) and in
any format (bottle opener, mAb-Fv, mAb-scFv, central-scFv,
bispecific mAb, central-Fv, one armed central-scFv, one armed
scFv-mAb, dual scFv, DVD-Ig or Trident). In particular they can be
combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.
[0104] FIG. 80A-80C depict a number of prior art sequences for Fvs
that bind human LAG-3 as vh and vl sequences. As will be
appreciated by those in the art, any of these Fvs can be combined
with an Fv that binds a costimulatory receptor (e.g. ICOS, GITR,
OX40 or 4-1BB, including the Fv sequences contained herein) and in
any format (bottle opener, mAb-Fv, mAb-scFv, central-scFv,
bispecific mAb, central-Fv, one armed central-scFv, one armed
scFv-mAb, dual scFv, DVD-Ig or Trident). In particular they can be
combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.
[0105] FIG. 81A-81C depict a number of prior art sequences for Fvs
that bind human TIM-3 as vh and vl sequences. As will be
appreciated by those in the art, any of these Fvs can be combined
with an Fv that binds a costimulatory receptor (e.g. ICOS, GITR,
OX40 or 4-1BB, including the Fv sequences contained herein) and in
any format (bottle opener, mAb-Fv, mAb-scFv, central-scFv,
bispecific mAb, central-Fv, one armed central-scFv, one armed
scFv-mAb, dual scFv, DVD-Ig or Trident). In particular they can be
combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.
[0106] FIG. 82 depict a number of prior art sequences for Fvs that
bind human BTLA as vh and vl sequences. As will be appreciated by
those in the art, any of these Fvs can be combined with an Fv that
binds a costimulatory receptor (e.g. ICOS, GITR, OX40 or 4-1BB,
including the Fv sequences contained herein) and in any format
(bottle opener, mAb-Fv, mAb-scFv, central-scFv, bispecific mAb,
central-Fv, one armed central-scFv, one armed scFv-mAb, dual scFv,
DVD-Ig or Trident). In particular they can be combined with
ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.
[0107] FIG. 83A-83D depict a number of prior art sequences for Fvs
that bind human TIGIT as vh and vl sequences. As will be
appreciated by those in the art, any of these Fvs can be combined
with an Fv that binds a costimulatory receptor (e.g. ICOS, GITR,
OX40 or 4-1BB, including the Fv sequences contained herein) and in
any format (bottle opener, mAb-Fv, mAb-scFv, central-scFv,
bispecific mAb, central-Fv, one armed central-scFv, one armed
scFv-mAb, dual scFv, DVD-Ig or Trident). In particular they can be
combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.
[0108] FIG. 84A-84C depict a number of BTLA ABDs, with additional
anti-BTLA ABDs being listed as SEQ ID NO: 3705-3736. The CDRs are
underlined, the scFv linker is double underlined (in the sequences,
the scFv linker is a positively charged scFv (GKPGS).sub.4 (SEQ ID
NO: 26209) linker, although as will be appreciated by those in the
art, this linker can be replaced by other linkers, including
uncharged or negatively charged linkers, some of which are depicted
in FIGS. 8A-B), and the slashes indicate the border(s) of the
variable domains. As above, the naming convention illustrates the
orientation of the scFv from N- to C-terminus; in the sequences
listed in this figure, they are all oriented as vh-scFv linker-vl
(from N- to C-terminus), although these sequences may also be used
in the opposite orientation, (from N- to C-terminus) vl-linker-vh.
As noted herein and is true for every sequence herein containing
CDRs, the exact identification of the CDR locations may be slightly
different depending on the numbering used as is shown in Table 1,
and thus included herein are not only the CDRs that are underlined
but also CDRs included within the vh and vl domains using other
numbering systems. Furthermore, as for all the sequences in the
Figures, these vh and vl sequences can be used either in a scFv
format or in a Fab format.
[0109] FIG. 85 is a matrix of possible combinations of the costim
and checkpoint ABDs, with all possible combinations possible. An
"A" in the box means that the PD-1 ABD is 1G6_L1.194_H1.279. A "B"
in the box means that the ICOS ABD is [ICOS]H.066_L0. A "C" in the
box means that the PD-1 is the scFv in the pair. A "D" in the box
means the CTLA-4 ABD is a Fab and is [CTLA-4]_H3_L0.22. An "E" in
the box means that the CTLA-4 ABD is a scFv and is
[CTLA-4]_H3.23_L0.22. An "F" in the box means that the LAG-3 ABD is
7G8_H3.30_L1.34. A "G" in the box means that the BTLA ABD is
9C6_H1.1_L1. An "H" in the box means that combination is a bottle
opener. An "I" in the box means the combination is Central-scFv
format.
[0110] FIG. 86 depicts two more ICOS.times.PD-1 bottle openers.
DETAILED DESCRIPTION OF THE INVENTION
I. Nomenclature
[0111] The bispecific antibodies of the invention are listed in
several different formats. Each polypeptide is given a unique
"XENP" number, although as will be appreciated in the art, a longer
sequence might contain a shorter one. For example, the heavy chain
of the scFv side monomer of a bottle opener format for a given
sequence will have a first XENP number, while the scFv domain will
have a different XENP number. Some molecules have three
polypeptides, so the XENP number, with the components, is used as a
name. Thus, the molecule XENP, which is in bottle opener format,
comprises three sequences, generally referred to as
"XENP23104-HC-Fab", XENP23104 HC-scFv" and "XENP23104 LC" or
equivalents, although one of skill in the art would be able to
identify these easily through sequence alignment. These XENP
numbers are in the sequence listing as well as identifiers, and
used in the Figures. In addition, one molecule, comprising the
three components, gives rise to multiple sequence identifiers. For
example, the listing of the Fab monomer has the full length
sequence, the variable heavy sequence and the three CDRs of the
variable heavy sequence; the light chain has a full length
sequence, a variable light sequence and the three CDRs of the
variable light sequence; and the scFv-Fc domain has a full length
sequence, an scFv sequence, a variable light sequence, 3 light
CDRs, a scFv linker, a variable heavy sequence and 3 heavy CDRs;
note that all molecules herein with a scFv domain use a single
charged scFv linker (+H), although others can be used. In addition,
the naming nomenclature of particular variable domains uses a
"Hx.xx_Ly.yy" type of format, with the numbers being unique
identifiers to particular variable chain sequences. Thus, the
variable domain of the scFv side of XENP23104 (which binds PD-1) is
"1G6_L1.194_H1.279", which indicates that the variable heavy domain
H1.279 was combined with the light domain L1.194. In the case that
these sequences are used as scFvs, the designation
"1G6_L1.194_H1.279", indicates that the variable heavy domain
H1.279 was combined with the light domain L1.194 and is in
vl-linker-vh orientation, from N- to C-terminus. This molecule with
the identical sequences of the heavy and light variable domains but
in the reverse order would be named "1G6_H1.279_L1.194". Similarly,
different constructs may "mix and match" the heavy and light chains
as will be evident from the sequence listing and the Figures.
II. Incorporation of Materials
A. Figures and Legends
[0112] Specifically incorporated by reference are the Figures,
Legends and Sequences from U.S. Ser. No. 62/479,723 and the
Figures, Legends and Sequences from U.S. Ser. No. 15/623,314. In
addition, the claims from U.S. Ser. No. 62/479,723 are additionally
specifically incorporated.
B. Sequences
[0113] Target Antigens: The sequence of human PD-1 (sp|Q15116) is
SEQ ID NO: 26226. The sequence of human PD-1, extracellular domain
(sp|Q15116|21-170) is SEQ ID NO: 26227. The sequence of Macaca
fascicularis PD-1 (tr|B0LAJ3) is SEQ ID NO: 26228. The sequence of
Macaca fascicularis PD-1, extracellular domain (predicted)
(tr|B0LAJ3|21-170) is SEQ ID NO: 26229. The sequence of human
CTLA-4 (sp|P16410) is SEQ ID NO: 26230. The sequence of human
CTLA-4, extracellular domain (sp|P16410.beta.6-161) is SEQ ID NO:
26231. The sequence of Macaca fascicularis CTLA-4 (tr|G7PL88) is
SEQ ID NO: 26232. The sequence of Macaca fascicularis CTLA-4,
extracellular domain (predicted) (tr|G7PL88) is SEQ ID NO: 26233.
The sequence of human LAG-3 (sp|P18627) is SEQ ID NO: 26234. The
sequence of human LAG-3, extracellular domain (sp|P18627|29-450) is
SEQ ID NO: 26235. The sequence of Macaca fascicularis LAG-3
(predicted) (gi|5444678151|ref|XP_005570011.1) is SEQ ID NO: 26236.
The sequence of Macaca fascicularis LAG-3, extracellular domain
(predicted) (gi|5444678151|ref|XP_005570011.1|29-450) is SEQ ID NO:
26237. The sequence of human TIM-3 (sp|Q8TDQ0) is SEQ ID NO: 26238.
The sequence of human TIM-3, extracellular domain
(sp|Q8TDQ0|22-202) is SEQ ID NO: 26239. The sequence of Macaca
fascicularis TIM-3 (predicted) (gi|355750365|gb|EHH54703.1) is SEQ
ID NO: 26240. The sequence of Macaca fascicularis TIM-3,
extracellular domain (predicted)
(gi|355750365|gb|EHH54703.1|22-203) is SEQ ID NO: 26241. The
sequence of human PD-L1 (sp|Q9NZQ7) is SEQ ID NO: 26242. The
sequence of human PD-L1, extracellular domain (sp|Q9NZQ7|19-238) is
SEQ ID NO: 26243. The sequence of Macaca fascicularis PD-L1
(predicted) (gb|XP_005581836.1) is SEQ ID NO: 26244. The sequence
of Macaca fascicularis PD-L1, extracellular domain (predicted)
(gb|XP_005581836.1|19-238) is SEQ ID NO: 26245. The sequence of
human ICOS (sp|Q9Y6W8) is SEQ ID NO: 26246. The sequence of human
ICOS, extracellular domain (sp|Q9Y6W8|21-140) is SEQ ID NO: 26247.
The sequence of Macaca fascicularis ICOS
(gi|544477053|ref|XP_005574075.1) is SEQ ID NO: 26248. The sequence
of Macaca fascicularis ICOS, extracellular domain (predicted)
(gi|544477053|ref|XP 005574075.1|21-140) is SEQ ID NO: 26249. The
sequence of human GITR (sp|Q9Y5U5) is SEQ ID NO: 26250. The
sequence of human GITR, extracellular domain (sp|Q9Y5U5|26-162) is
SEQ ID NO: 26251. The sequence of Macaca fascicularis GITR
(predicted) (ref|XP_005545180.1) is SEQ ID NO: 26252. The sequence
of Macaca fascicularis GITR, extracellular domain (predicted)
(ref|XP_005545180.1|26-162) is SEQ ID NO: 26253. The sequence of
human OX40 (sp|P43489) is SEQ ID NO: 26254. The sequence of human
OX40, extracellular domain (sp|P43489|29-214) is SEQ ID NO: 26255.
The sequence of Macaca fascicularis OX40 (predicted)
(ref|XP_005545179.1) is SEQ ID NO: 26256. The sequence of Macaca
fascicularis OX40, extracellular domain (predicted)
(ref|XP_005545179.1|29-214) is SEQ ID NO: 26257. The sequence of
human 4-1BB (sp|Q07011) is SEQ ID NO: 26258. The sequence of human
4-1BB, extracellular domain (sp|Q0701.parallel.24-186) is SEQ ID
NO: 26259. The sequence of Macaca fascicularis 4-1BB (predicted)
(ref|XP_005544945.1) is SEQ ID NO: 26260. The sequence of Macaca
fascicularis 4-1BB, extracellular domain (predicted)
(ref|XP_005544945.1|24-186) is SEQ ID NO: 26261.
[0114] ICOS binding domains: In addition the sequences shown in
FIG. 19, FIGS. 20A-20G and FIG. 24, SEQ ID NO:27869-28086 contain a
number of ICOS Fab sequences (heavy chain VH1-CH1 and light chain
VL1-CL) as indicated in the naming nomenclature. Reference for the
CDRs and for the junction between the variable junctions is shown
in FIG. 40 of U.S. Ser. No. 62/479,723 (hereby incorporated by
reference as well as the Legend), although from the SEQ listing one
of skill in the art will be able to ascertain the CDRs (see Table 1
for numbering and/or through sequence alignment) as well as for the
junctions (e.g. heavy chain CH1 generally starts with the sequence
"ASTK . . . " and light chain constant domain generally starts with
"RTVA . . . ". SEQ ID NO:28087-28269 show the three sequences for
"one armed mAb" (FIG. 2N; Fab-Fc, Fc only and light chain) as shown
in the naming nomenclature. Reference for the CDRs and for the
junction between the variable junctions is shown in FIG. 41 of U.S.
Ser. No. 62/479,723 (hereby incorporated by reference as well as
the Legend), although from the SEQ listing one of skill in the art
will be able to ascertain the CDRs (see Table 1 for numbering
and/or through sequence alignment) as well as for the junctions
(e.g. heavy chain CH1 generally starts with the sequence "ASTK . .
. " and light chain constant domain generally starts with "RTVA . .
. ". Additional one armed ICOS molecules are shown in FIGS.
68A-68G. SEQ ID NO:28549-28556 show some control antibodies (HC and
LC) from which the Fvs can be used as ICOS ABDs as well; reference
for the CDRs and for the junction between the variable junctions is
shown in FIG. 44 of U.S. Ser. No. 62/479,723 (hereby incorporated
by reference as well as the Legend), although from the SEQ listing
one of skill in the art will be able to ascertain the CDRs (see
Table 1 for numbering and/or through sequence alignment) as well as
for the junctions (e.g. heavy chain CH1 generally starts with the
sequence "ASTK . . . " and light chain constant domain generally
starts with "RTVA . . . ". SEQ ID NO:28557-28665 show some ICOS
scFvs that find use in combination in the invention; reference for
the CDRs and for the junction between the variable junctions is
shown in FIG. 45 of U.S. Ser. No. 62/479,723 (hereby incorporated
by reference as well as the Legend), although from the SEQ listing
one of skill in the art will be able to ascertain the CDRs (see
Table 1 for numbering and/or through sequence alignment) as well as
for the junctions, as the scFvs utilize the charged linker
(GKPGS).sub.4 between the vh and vl domains. Thus, suitable ICOS
ABDs for use in combination with ABDs for checkpoint receptors are
shown in FIG. 19, FIGS. 20A-20G, FIG. 24, FIGS. 68A-68G and FIGS.
77A-77BA-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665, and [ICOS]_H0.66_L0 and
[ICOS]_H0_L0.
[0115] In addition, suitable ICOS.times.PD-1 bottle opener
sequences include those in FIG. 3 as well as those in SEQ ID
NO:28270-28548, which correspond to the sequences depicted in FIGS.
42 and 43 of U.S. Ser. No. 62/479,723 (both of which are hereby
incorporated by reference as well as the Legends and sequences
therein), again using these figures to show CDRs, junctions,
etc.
III. Overview
[0116] Therapeutic antibodies directed against immune
immunomodulatory inhibitors such as PD-1 are showing great promise
in limited circumstances in the clinic for the treatment of cancer.
Cancer can be considered as an inability of the patient to
recognize and eliminate cancerous cells. In many instances, these
transformed (e.g. cancerous) cells counteract immunosurveillance.
There are natural control mechanisms that limit T-cell activation
in the body to prevent unrestrained T-cell activity, which can be
exploited by cancerous cells to evade or suppress the immune
response. Restoring the capacity of immune effector
cells-especially T cells--to recognize and eliminate cancer is the
goal of immunotherapy. The field of immuno-oncology, sometimes
referred to as "immunotherapy" is rapidly evolving, with several
recent approvals of T cell checkpoint inhibitory antibodies such as
YERVOY.RTM., KEYTRUDA.RTM. and OPDIVO.RTM.. These antibodies are
generally referred to as "checkpoint inhibitors" because they block
normally negative regulators of T cell immunity. It is generally
understood that a variety of immunomodulatory signals, both
costimulatory and coinhibitory, can be used to orchestrate an
optimal antigen-specific immune response.
[0117] Checkpoint inhibitor monoclonal antibodies bind to
immunomodulatory inhibitor proteins such as PD-1, which under
normal circumstances prevent or suppress activation of cytotoxic T
cells (CTLs). By inhibiting the immunomodulatory protein, for
example through the use of antibodies that bind these proteins, an
increased T cell response against tumors can be achieved. That is,
these cancer immunomodulatory proteins suppress the immune
response; when the proteins are blocked, for example using
antibodies to the immunomodulatory protein, the immune system is
activated, leading to immune stimulation, resulting in treatment of
conditions such as cancer and infectious disease. Antibodies to
either the PD-1 protein or its binding partner, PD-L1, leads to T
cell activation.
[0118] Another area of current interest for harnessing the
patient's immune system to fight disease involves the
co-stimulation of T cells using agonistic antibodies that bind to
co-stimulatory proteins such as ICOS (Inducible T cell
Co-Stimulator, also referred to as CD278) which adds a positive
signal to overcome the negative signaling of the immune checkpoint
proteins on the T-cells. ICOS is a type I transmembrane protein
comprising an extracellular (Ig) V-like domain, and serves as the
receptor for the B7h co-stimulatory molecule.
[0119] Recent work shows that some tumor infiltrating lymphocytes
(TILs) co-express PD-1 and ICOS (see Gros, J. Clinical Invest.
124(5):2246 (2014)).
[0120] Bispecific antibodies, which can bind two different targets
simultaneously, offer the potential to improve the selectivity of
targeting TILs vs peripheral T cells, while also reducing cost of
therapy. The bivalent interaction of an antibody with two targets
on a cell surface should--in some cases--lead to a higher binding
avidity relative to a monovalent interaction with one target at a
time. Because of this, normal bivalent antibodies tend to have high
avidity for their target on a cell surface. With bispecific
antibodies, the potential exists to create higher selectivity for
cells that simultaneously express two different targets, utilizing
the higher avidity afforded by simultaneous binding to both
targets.
[0121] Accordingly, the present invention provides bispecific
immunomodulatory antibodies, that bind to cells expressing the two
antigens and methods of activating T cells and/or NK cells to treat
diseases such as cancer and infectious diseases, and other
conditions where increased immune activity results in
treatment.
[0122] Thus, the invention is directed, in some instances, to
solving the issue of toxicity and expense of administering multiple
antibodies by providing bispecific antibodies that bind to two
different immunomodulatory molecules (one a checkpoint receptor and
the other a costimulatory receptor) on a single cell and
advantageously requiring administration of only one therapeutic
substance.
[0123] Bispecific antibodies offer the opportunity to combine
immune immunomodulatory blockade with costimulation in one
molecule. However, it is not obvious what combination of immune
immunomodulatory plus costimulatory protein or what binding
stoichiometry (monovalent+monovalent, monovalent+bivalent, etc.)
would be efficacious. Here we identify bispecific antibodies that
binding monovalently to a costimulatory protein (such as ICOS) and
monovalent binding to a checkpoint receptor (such as PD-1) that are
capable of inducing robust T cell activation.
[0124] Surprisingly, while conventional wisdom states that
monovalent antibodies do not result in agonism, the present work
shows the unexpected results of agonism of the ICOS receptor with
the monovalent bispecific antibodies of the invention. See Merchant
et al., PNAS Jul. 23 2013 E2987-E2996, "[w]hile initial screening
of bivalent antibodies produced agonists of MET, engineering them
into monovalent antibodies produces antagonists instead." This
positive result with only monovalent binding to ICOS is unexpected
because it is thought that at least bivalent binding to a
costimulatory protein is necessary to provide the required level of
receptor clustering on the cell surface for triggering signaling.
As shown by Fos et al., J. Immunol. 2008:1969-1977, ICOS ligation
induces AKT phosphorylation. The studies here in use AKT
phosphorylation as an indicator of ICOS agonism, and this effect is
seen for both "one armed ICOS" (see Example 5A(a)) and for
bispecific antibodies that bind ICOS monovalently. As shown in
Example 7 and in FIG. 70, the one-arm XENP20266 that only binds
ICOS monovalently promotes more AKT phosphorylation than XENP16435,
which binds ICOS bivalently (e.g. as a traditional mAb).
[0125] Accordingly, the present invention is directed to novel
constructs to provide heterodimeric, bispecific antibodies that
allow binding to a checkpoint receptor as well as human ICOS.
[0126] Note that generally these bispecific antibodies are named
"anti-PD-1.times.anti-ICOS", or generally simplistically or for
ease (and thus interchangeably) as "PD-1.times.ICOS", etc. for each
pair.
[0127] The heterodimeric bispecific immunomodulatory antibodies of
the invention are useful to treat a variety of types of cancers. As
will be appreciated by those in the art, in contrast to traditional
monoclonal antibodies that bind to tumor antigens, or to the newer
classes of bispecific antibodies that bind, for example, CD3 and
tumor antigens (such as described in U.S. Ser. No. 15/141,350, for
example), immunomodulatory antibodies are used to increase the
immune response but are not generally tumor specific in their
action. That is, the bispecific immunomodulatory antibodies of the
invention inhibit the suppression of the immune system, generally
leading to T cell activation, which in turn leads to greater immune
response to cancerous cells and thus treatment.
[0128] As discussed below, there are a variety of ways that T cell
activation can be measured. Functional effects of the bispecific
immunomodulatory antibodies on NK and T-cells can be assessed in
vitro (and in some cases in vivo, as described more fully below) by
measuring changes in the following parameters: proliferation,
cytokine release and cell-surface makers. For NK cells, increases
in cell proliferation, cytotoxicity (ability to kill target cells
as measured by increases in CD107a, granzyme, and perforin
expression, or by directly measuring target cells killing),
cytokine production (e.g. IFN-.gamma. and TNF), and cell surface
receptor expression (e.g. CD25) is indicative of immune modulation,
e.g. enhanced killing of cancer cells. For T-cells, increases in
proliferation, increases in expression of cell surface markers of
activation (e.g. CD25, CD69, CD137, and PD1), cytotoxicity (ability
to kill target cells), and cytokine production (e.g. IL-2, IL-4,
IL-6, IFN, TNF-.alpha., IL-10, IL-17A) are indicative of immune
modulation, e.g. enhanced killing of cancer cells. Accordingly,
assessment of treatment can be done using assays that evaluate one
or more of the following: (i) increases in immune response, (ii)
increases in activation of .alpha..beta. and/or .gamma..delta. T
cells, (iii) increases in cytotoxic T cell activity, (iv) increases
in NK and/or NKT cell activity, (v) alleviation of .alpha..beta.
and/or .gamma..delta. T-cell suppression, (vi) increases in
pro-inflammatory cytokine secretion, (vii) increases in IL-2
secretion; (viii) increases in interferon-.gamma. production, (ix)
increases in Th1 response, (x) decreases in Th2 response, (xi)
decreases or eliminates cell number and/or activity of at least one
of regulatory T cells, (xii) increases in IL-2 secretion.
[0129] Thus, in some embodiments the invention provides the use of
bispecific immunomodulatory antibodies to perform one or more of
the following in a subject in need thereof: (a) upregulating
pro-inflammatory cytokines; (b) increasing T-cell proliferation
and/or expansion; (c) increasing interferon-.gamma. or TNF-.alpha.
production by T-cells; (d) increasing IL-2 secretion; (e)
stimulating antibody responses; (f) inhibiting cancer cell growth;
(g) promoting antigenic specific T cell immunity; (h) promoting
CD4+ and/or CD8+ T cell activation; (i) alleviating T-cell
suppression; (j) promoting NK cell activity; (k) promoting
apoptosis or lysis of cancer cells; and/or (l) cytotoxic or
cytostatic effect on cancer cells.
[0130] Accordingly, the present invention provides bispecific
immunomodulatory antibodies. There are a number of formats that can
be used in the present invention, as generally shown in FIG. 2,
many of which are heterodimeric (although not all, as DVD-Ig, for
example).
[0131] The heterodimeric antibodies constructs are based on the
self-assembling nature of the two Fc domains of the heavy chains of
antibodies, e.g. two "monomers" that assemble into a "dimer".
Heterodimeric antibodies are made by altering the amino acid
sequence of each monomer as more fully discussed below. Thus, the
present invention is generally directed to the creation of
heterodimeric antibodies, which can co-engage two antigens in
several ways, relying on amino acid variants in the constant
regions that are different on each chain to promote heterodimeric
formation and/or allow for ease of purification of heterodimers
over the homodimers.
[0132] Thus, the present invention provides bispecific
immunomodulatory antibodies. An ongoing problem in antibody
technologies is the desire for "bispecific" antibodies that bind to
two (or more) different antigens simultaneously, in general thus
allowing the different antigens to be brought into proximity and
resulting in new functionalities and new therapies. In general,
these antibodies are made by including genes for each heavy and
light chain into the host cells (generally, in the present
invention, genes for two heavy chain monomers and a light chain as
outlined herein). This generally results in the formation of the
desired heterodimer (A-B), as well as the two homodimers (A-A and
B-B). However, a major obstacle in the formation of bispecific
antibodies is the difficulty in purifying the heterodimeric
antibodies away from the homodimeric antibodies and/or biasing the
formation of the heterodimer over the formation of the
homodimers.
[0133] To solve this issue, there are a number of mechanisms that
can be used to generate the heterodimers of the present invention.
In addition, as will be appreciated by those in the art, these
mechanisms can be combined to ensure high heterodimerization. Thus,
amino acid variants that lead to the production of heterodimeric
antibodies are referred to as "heterodimerization variants". As
discussed below, heterodimerization variants can include steric
variants (e.g. the "knobs and holes" or "skew" variants described
below and the "charge pairs" variants described below) as well as
"pI variants", which allows purification of homodimers away from
heterodimers.
[0134] One mechanism is generally referred to in the art as "knobs
and holes" ("KIH") or sometimes herein as "skew" variants,
referring to amino acid engineering that creates steric influences
to favor heterodimeric formation and disfavor homodimeric formation
can also optionally be used; this is sometimes referred to as
"knobs and holes"; as described in Ridgway et al., Protein
Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997
270:26; U.S. Pat. No. 8,216,805, US 2012/0149876, all of which are
hereby incorporated by reference in their entirety. The Figures
identify a number of "monomer A-monomer B" pairs that include
"knobs and holes" amino acid substitutions. In addition, as
described in Merchant et al., Nature Biotech. 16:677 (1998), these
"knobs and hole" mutations can be combined with disulfide bonds to
skew formation to heterodimerization. Of use in the present
invention are T366S/L368A/Y407V paired with T366W, as well as this
variant with a bridging disulfide, T366S/L368A/Y407V/Y349C paired
with T366W/S354C, particularly in combination with other
heterodimerization variants including pI variants as outlined
below.
[0135] An additional mechanism that finds use in the generation of
heterodimeric antibodies is sometimes referred to as "electrostatic
steering" or "charge pairs" as described in Gunasekaran et al., J.
Biol. Chem. 285(25):19637 (2010), hereby incorporated by reference
in its entirety. This is sometimes referred to herein as "charge
pairs". In this embodiment, electrostatics are used to skew the
formation towards heterodimerization. As those in the art will
appreciate, these may also have an effect on pI, and thus on
purification, and thus could in some cases also be considered pI
variants. However, as these were generated to force
heterodimerization and were not used as purification tools, they
are classified as "steric variants". These include, but are not
limited to, D221E/P228E/L368E paired with D221R/P228R/K409R (e.g.
these are "monomer corresponding sets) and C220E/P228E/368E paired
with C220R/E224R/P228R/K409R and others shown in the Figures.
[0136] In the present invention, in some embodiments, pI variants
are used to alter the pI of one or both of the monomers and thus
allowing the isoelectric purification of A-A, A-B and B-B dimeric
proteins.
[0137] In the present invention, there are several basic mechanisms
that can lead to ease of purifying heterodimeric proteins; one
relies on the use of pI variants, such that each monomer has a
different pI, thus allowing the isoelectric purification of A-A,
A-B and B-B dimeric proteins. Alternatively, some scaffold formats,
such as the "triple F" or "bottle opener" format, also allows
separation on the basis of size. As is further outlined below, it
is also possible to "skew" the formation of heterodimers over
homodimers. Thus, a combination of steric heterodimerization
variants and pI or charge pair variants find particular use in the
invention. Additionally, as more fully outlined below, scaffolds
that utilize scFv(s) such as the Triple F format can include
charged scFv linkers (either positive or negative), that give a
further pI boost for purification purposes. As will be appreciated
by those in the art, some Triple F formats are useful with just
charged scFv linkers and no additional pI adjustments, although the
invention does provide the use of skew variants with charged scFv
linkers as well (and combinations of Fc, FcRn and KO variants
discussed herein).
[0138] In the present invention that utilizes pI as a separation
mechanism to allow the purification of heterodimeric proteins,
amino acid variants can be introduced into one or both of the
monomer polypeptides; that is, the pI of one of the monomers
(referred to herein for simplicity as "monomer A") can be
engineered away from monomer B, or both monomer A and B change be
changed, with the pI of monomer A increasing and the pI of monomer
B decreasing. As is outlined more fully below, the pI changes of
either or both monomers can be done by removing or adding a charged
residue (e.g. a neutral amino acid is replaced by a positively or
negatively charged amino acid residue, e.g. glycine to glutamic
acid), changing a charged residue from positive or negative to the
opposite charge (aspartic acid to lysine) or changing a charged
residue to a neutral residue (e.g. loss of a charge; lysine to
serine). A number of these variants are shown in the Figures. In
addition, suitable pI variants for use in the creation of
heterodimeric antibodies herein are those that are isotypic, e.g.
importing pI variants from different IgG isotypes such that pI is
changed without introducing significant immunogenicity; see FIG. 29
from US Publication No. 20140288275, hereby incorporated by
reference in its entirety.
[0139] Accordingly, in this embodiment of the present invention
provides for creating a sufficient change in pI in at least one of
the monomers such that heterodimers can be separated from
homodimers. As will be appreciated by those in the art, and as
discussed further below, this can be done by using a "wild type"
heavy chain constant region and a variant region that has been
engineered to either increase or decrease its pI (wt A-+B or wt
A--B), or by increasing one region and decreasing the other region
(A+-B- or A-B+).
[0140] Thus, in general, a component of some embodiments of the
present invention are amino acid variants in the constant regions
of antibodies that are directed to altering the isoelectric point
(pI) of at least one, if not both, of the monomers of a dimeric
protein to form "pI heterodimers" (when the protein is an antibody,
these are referred to as "pI antibodies") by incorporating amino
acid substitutions ("pI variants" or "pI substitutions") into one
or both of the monomers. As shown herein, the separation of the
heterodimers from the two homodimers can be accomplished if the pIs
of the two monomers differ by as little as 0.1 pH unit, with 0.2,
0.3, 0.4 and 0.5 or greater all finding use in the present
invention.
[0141] As will be appreciated by those in the art, the number of pI
variants to be included on each or both monomer(s) to get good
separation will depend in part on the starting pI of the scFv and
Fab of interest. That is, to determine which monomer to engineer or
in which "direction" (e.g. more positive or more negative), the Fv
sequences of the two target antigens are calculated and a decision
is made from there. As is known in the art, different Fvs will have
different starting pIs which are exploited in the present
invention. In general, as outlined herein, the pIs are engineered
to result in a total pI difference of each monomer of at least
about 0.1 logs, with 0.2 to 0.5 being preferred as outlined herein.
Furthermore, as will be appreciated by those in the art and
outlined herein, in some cases (depending on the format)
heterodimers can be separated from homodimers on the basis of size
(e.g. Molecular weight). For example, as shown in some embodiments
of FIG. 2, some formats result in homodimers and heterodimers with
different sizes (e.g. for bottle openers, one homodimer is a "dual
scFv" format, one homodimer is a standard antibody, and the
heterodimer has one Fab and one scFv).
[0142] In the case where pI variants are used to achieve purified
heterodimers over homodimers, by using the constant region(s) of
the heavy chain(s), a more modular approach to designing and
purifying multispecific proteins, including antibodies, is
provided. Thus, in some embodiments, heterodimerization variants
(including skew and purification heterodimerization variants) are
not included in the variable regions, such that each individual
antibody must be engineered. In addition, in some embodiments, the
possibility of immunogenicity resulting from the pI variants is
significantly reduced by importing pI variants from different IgG
isotypes such that pI is changed without introducing significant
immunogenicity. Thus, an additional problem to be solved is the
elucidation of low pI constant domains with high human sequence
content, e.g. the minimization or avoidance of non-human residues
at any particular position.
[0143] A side benefit that can occur with this pI engineering is
also the extension of serum half-life and increased FcRn binding.
That is, as described in U.S. Ser. No. 13/194,904 (incorporated by
reference in its entirety), lowering the pI of antibody constant
domains (including those found in antibodies and Fc fusions) can
lead to longer serum retention in vivo. These pI variants for
increased serum half life also facilitate pI changes for
purification.
[0144] In addition, it should be noted that the pI variants of the
heterodimerization variants give an additional benefit for the
analytics and quality control process of bispecific antibodies, as
the ability to either eliminate, minimize and distinguish when
homodimers are present is significant. Similarly, the ability to
reliably test the reproducibility of the heterodimeric protein
production is important.
[0145] First and second antigens of the invention are herein
referred to as antigen-1 and antigen-2 respectively, with one being
a costimulatory receptor and one being a checkpoint receptor. One
heterodimeric scaffold that finds particular use in the present
invention is the "triple f" or "bottle opener" scaffold format. In
this embodiment, one heavy chain of the antibody contains an single
chain fv ("scfv", as defined below) and the other heavy chain is a
"regular" fab format, comprising a variable heavy chain and a light
chain. This structure is sometimes referred to herein as "triple f"
format (scfv-fab-fc) or the "bottle-opener" format, due to a rough
visual similarity to a bottle-opener (see FIG. 2). The two chains
are brought together by the use of amino acid variants in the
constant regions (e.g. the Fc domain and/or the hinge region) that
promote the formation of heterodimeric antibodies as is described
more fully below.
[0146] There are several distinct advantages to the present "triple
F" format. As is known in the art, antibody analogs relying on two
scFv constructs often have stability and aggregation problems,
which can be alleviated in the present invention by the addition of
a "regular" heavy and light chain pairing. In addition, as opposed
to formats that rely on two heavy chains and two light chains,
there is no issue with the incorrect pairing of heavy and light
chains (e.g. heavy 1 pairing with light 2, etc.)
[0147] Furthermore, as outlined herein, additional amino acid
variants may be introduced into the bispecific antibodies of the
invention, to add additional functionalities. For example, amino
acid changes within the Fc region can be added (either to one
monomer or both) to facilitate increased ADCC or CDC (e.g. altered
binding to Fc.gamma. receptors) as well as to increase binding to
FcRn and/or increase serum half-life of the resulting molecules. As
is further described herein and as will be appreciated by those in
the art, any and all of the variants outlined herein can be
optionally and independently combined with other variants.
[0148] Similarly, another category of functional variants are
"Fc.gamma. ablation variants" or "Fc knock out (FcKO or KO)
variants. In these embodiments, for some therapeutic applications,
it is desirable to reduce or remove the normal binding of the Fc
domain to one or more or all of the Fc.gamma. receptors (e.g.
Fc.gamma.R1, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIIa, etc.) to
avoid additional mechanisms of action. That is, for example, it is
generally desirable to ablate Fc.gamma.RIIIa binding to eliminate
or significantly reduce ADCC activity. Suitable ablation variants
are shown in FIG. 6.
IV. Definitions
[0149] In order that the application may be more completely
understood, several definitions are set forth below. Such
definitions are meant to encompass grammatical equivalents.
[0150] By "ablation" herein is meant a decrease or removal of
activity. Thus for example, "ablating Fc.gamma.R binding" means the
Fc region amino acid variant has less than 50% starting binding as
compared to an Fc region not containing the specific variant, with
less than 70-80-90-95-98% loss of activity being preferred, and in
general, with the activity being below the level of detectable
binding in a BIACORE.RTM. assay. Of particular use in the ablation
of Fc.gamma.R binding are those shown in FIG. 6.
[0151] By "ADCC" or "antibody dependent cell-mediated cytotoxicity"
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. ADCC is correlated with binding to Fc.gamma.RIIIa;
increased binding to Fc.gamma.RIIIa leads to an increase in ADCC
activity. As is discussed herein, many embodiments of the invention
ablate ADCC activity entirely.
[0152] By "ADCP" or antibody dependent cell-mediated phagocytosis
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause phagocytosis
of the target cell.
[0153] By "antigen binding domain" or "ABD" herein is meant a set
of six Complementary Determining Regions (CDRs) that, when present
as part of a polypeptide sequence, specifically binds a target
antigen as discussed herein. Thus, a "immunomodulatory antigen
binding domain" binds a target immunomodulatory antigen as outlined
herein. As is known in the art, these CDRs are generally present as
a first set of variable heavy CDRs (vhCDRs or VHCDRs) and a second
set of variable light CDRs (vlCDRs or V.sub.LCDR5), each comprising
three CDRs: vhCDR1, vhCDR2, vhCDR3 for the heavy chain and vlCDR1,
vlCDR2 and vlCDR3 for the light. The CDRs are present in the
variable heavy and variable light domains, respectively, and
together form an Fv region. Thus, in some cases, the six CDRs of
the antigen binding domain are contributed by a variable heavy and
variable light chain. In a "Fab" format, the set of 6 CDRs are
contributed by two different polypeptide sequences, the variable
heavy domain (vh or VII; containing the vhCDR1, vhCDR2 and vhCDR3)
and the variable light domain (vl or V.sub.L; containing the
vlCDR1, vlCDR2 and vlCDR3), with the C-terminus of the vh domain
being attached to the N-terminus of the CH1 domain of the heavy
chain and the C-terminus of the vl domain being attached to the
N-terminus of the constant light domain (and thus forming the light
chain). In a scFv format, the vh and vl domains are covalently
attached, generally through the use of a linker as outlined herein,
into a single polypeptide sequence, which can be either (starting
from the N-terminus) vh-linker-vl or vl-linker-vh, with the former
being generally preferred (including optional domain linkers on
each side, depending on the format used.
[0154] By "modification" herein is meant an amino acid
substitution, insertion, and/or deletion in a polypeptide sequence
or an alteration to a moiety chemically linked to a protein. For
example, a modification may be an altered carbohydrate or PEG
structure attached to a protein. By "amino acid modification"
herein is meant an amino acid substitution, insertion, and/or
deletion in a polypeptide sequence. For clarity, unless otherwise
noted, the amino acid modification is always to an amino acid coded
for by DNA, e.g. the 20 amino acids that have codons in DNA and
RNA.
[0155] By "amino acid substitution" or "substitution" herein is
meant the replacement of an amino acid at a particular position in
a parent polypeptide sequence with a different amino acid. In
particular, in some embodiments, the substitution is to an amino
acid that is not naturally occurring at the particular position,
either not naturally occurring within the organism or in any
organism. For example, the substitution E272Y refers to a variant
polypeptide, in this case an Fc variant, in which the glutamic acid
at position 272 is replaced with tyrosine. For clarity, a protein
which has been engineered to change the nucleic acid coding
sequence but not change the starting amino acid (for example
exchanging CGG (encoding arginine) to CGA (still encoding arginine)
to increase host organism expression levels) is not an "amino acid
substitution"; that is, despite the creation of a new gene encoding
the same protein, if the protein has the same amino acid at the
particular position that it started with, it is not an amino acid
substitution.
[0156] By "amino acid insertion" or "insertion" as used herein is
meant the addition of an amino acid sequence at a particular
position in a parent polypeptide sequence. For example, -233E or
233E designates an insertion of glutamic acid after position 233
and before position 234. Additionally, -233ADE or A233ADE
designates an insertion of AlaAspGlu after position 233 and before
position 234.
[0157] By "amino acid deletion" or "deletion" as used herein is
meant the removal of an amino acid sequence at a particular
position in a parent polypeptide sequence. For example, E233- or
E233#, E233( ) or E233del designates a deletion of glutamic acid at
position 233. Additionally, EDA233- or EDA233# designates a
deletion of the sequence GluAspAla that begins at position 233.
[0158] By "variant protein" or "protein variant", or "variant" as
used herein is meant a protein that differs from that of a parent
protein by virtue of at least one amino acid modification. Protein
variant may refer to the protein itself, a composition comprising
the protein, or the amino sequence that encodes it. Preferably, the
protein variant has at least one amino acid modification compared
to the parent protein, e.g. from about one to about seventy amino
acid modifications, and preferably from about one to about five
amino acid modifications compared to the parent. As described
below, in some embodiments the parent polypeptide, for example an
Fc parent polypeptide, is a human wild type sequence, such as the
Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequences
with variants can also serve as "parent polypeptides", for example
the IgG1/2 hybrid can be included. The protein variant sequence
herein will preferably possess at least about 80% identity with a
parent protein sequence, and most preferably at least about 90%
identity, more preferably at least about 95-98-99% identity.
Variant protein can refer to the variant protein itself,
compositions comprising the protein variant, or the DNA sequence
that encodes it.
[0159] Accordingly, by "antibody variant" or "variant antibody" as
used herein is meant an antibody that differs from a parent
antibody by virtue of at least one amino acid modification, "IgG
variant" or "variant IgG" as used herein is meant an antibody that
differs from a parent IgG (again, in many cases, from a human IgG
sequence) by virtue of at least one amino acid modification, and
"immunoglobulin variant" or "variant immunoglobulin" as used herein
is meant an immunoglobulin sequence that differs from that of a
parent immunoglobulin sequence by virtue of at least one amino acid
modification. "Fc variant" or "variant Fc" as used herein is meant
a protein comprising an amino acid modification in an Fc domain.
The Fc variants of the present invention are defined according to
the amino acid modifications that compose them. Thus, for example,
N434S or 434S is an Fc variant with the substitution serine at
position 434 relative to the parent Fc polypeptide, wherein the
numbering is according to the EU index. Likewise, M428L/N434S
defines an Fc variant with the substitutions M428L and N434S
relative to the parent Fc polypeptide. The identity of the WT amino
acid may be unspecified, in which case the aforementioned variant
is referred to as 428L/434S. It is noted that the order in which
substitutions are provided is arbitrary, that is to say that, for
example, 428L/434S is the same Fc variant as M428L/N434S, and so
on. For all positions discussed in the present invention that
relate to antibodies, unless otherwise noted, amino acid position
numbering is according to the EU index. The EU index or EU index as
in Kabat or EU numbering scheme refers to the numbering of the EU
antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,
hereby entirely incorporated by reference.) The modification can be
an addition, deletion, or substitution. Substitutions can include
naturally occurring amino acids and, in some cases, synthetic amino
acids. Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO
03/073238; US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W.
Chin et al., (2002), Journal of the American Chemical Society
124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem
11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of
America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002),
Chem. 1-10, all entirely incorporated by reference.
[0160] As used herein, "protein" herein is meant at least two
covalently attached amino acids, which includes proteins,
polypeptides, oligopeptides and peptides. The peptidyl group may
comprise naturally occurring amino acids and peptide bonds, or
synthetic peptidomimetic structures, i.e. "analogs", such as
peptoids (see Simon et al., PNAS USA 89(20):9367 (1992), entirely
incorporated by reference). The amino acids may either be naturally
occurring or synthetic (e.g. not an amino acid that is coded for by
DNA); as will be appreciated by those in the art. For example,
homo-phenylalanine, citrulline, ornithine and noreleucine are
considered synthetic amino acids for the purposes of the invention,
and both D- and L-(R or S) configured amino acids may be utilized.
The variants of the present invention may comprise modifications
that include the use of synthetic amino acids incorporated using,
for example, the technologies developed by Schultz and colleagues,
including but not limited to methods described by Cropp &
Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004,
Proc Natl Acad Sci USA 101 (2):7566-71, Zhang et al., 2003,
303(5656):371-3, and Chin et al., 2003, Science 301(5635):964-7,
all entirely incorporated by reference. In addition, polypeptides
may include synthetic derivatization of one or more side chains or
termini, glycosylation, PEGylation, circular permutation,
cyclization, linkers to other molecules, fusion to proteins or
protein domains, and addition of peptide tags or labels.
[0161] By "residue" as used herein is meant a position in a protein
and its associated amino acid identity. For example, Asparagine 297
(also referred to as Asn297 or N297) is a residue at position 297
in the human antibody IgG1.
[0162] By "Fab" or "Fab region" as used herein is meant the
polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin
domains. Fab may refer to this region in isolation, or this region
in the context of a full length antibody, antibody fragment or Fab
fusion protein.
[0163] By "Fv" or "Fv fragment" or "Fv region" as used herein is
meant a polypeptide that comprises the VL and VH domains of a
single ABD. As will be appreciated by those in the art, these
generally are made up of two chains, or can be combined (generally
with a linker as discussed herein) to form an scFv. In some cases,
for example in the "central-Fv" and "DVD-Ig" formats, an "extra" vh
and vl domain is added that serves as a scFv but where the vh and
vl domains are not linked using a scFv linker between them.
[0164] By "single chain Fv" or "scFv" herein is meant a variable
heavy domain covalently attached to a variable light domain,
generally using a scFv linker as discussed herein, to form a scFv
or scFv domain. A scFv domain can be in either orientation from N-
to C-terminus (vh-linker-vl or vl-linker-vh).
[0165] By "IgG subclass modification" or "isotype modification" as
used herein is meant an amino acid modification that converts one
amino acid of one IgG isotype to the corresponding amino acid in a
different, aligned IgG isotype. For example, because IgG1 comprises
a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y
substitution in IgG2 is considered an IgG subclass
modification.
[0166] By "non-naturally occurring modification" as used herein is
meant an amino acid modification that is not isotypic. For example,
because none of the IgGs comprise a serine at position 434, the
substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof)
is considered a non-naturally occurring modification.
[0167] By "amino acid" and "amino acid identity" as used herein is
meant one of the 20 naturally occurring amino acids that are coded
for by DNA and RNA.
[0168] By "effector function" as used herein is meant a biochemical
event that results from the interaction of an antibody Fc region
with an Fc receptor or ligand. Effector functions include but are
not limited to ADCC, ADCP, and CDC.
[0169] By "IgG Fc ligand" as used herein is meant a molecule,
preferably a polypeptide, from any organism that binds to the Fc
region of an IgG antibody to form an Fc/Fc ligand complex. Fc
ligands include but are not limited to Fc.gamma.RIs, Fc.gamma.RIIs,
Fc.gamma.RIIIs, FcRn, C1q, C3, mannan binding lectin, mannose
receptor, staphylococcal protein A, streptococcal protein G, and
viral Fc.gamma.R. Fc ligands also include Fc receptor homologs
(FcRH), which are a family of Fc receptors that are homologous to
the Fc.gamma.Rs (Davis et al., 2002, Immunological Reviews
190:123-136, entirely incorporated by reference). Fc ligands may
include undiscovered molecules that bind Fc. Particular IgG Fc
ligands are FcRn and Fc gamma receptors. By "Fc ligand" as used
herein is meant a molecule, preferably a polypeptide, from any
organism that binds to the Fc region of an antibody to form an
Fc/Fc ligand complex.
[0170] By "Fc gamma receptor", "Fc.gamma.R" or "FcgammaR" as used
herein is meant any member of the family of proteins that bind the
IgG antibody Fc region and is encoded by an Fc.gamma.R gene. In
humans this family includes but is not limited to Fc.gamma.RI
(CD64), including isoforms Fc.gamma.RIa, Fc.gamma.RIb, and
Fc.gamma.RIc; Fc.gamma.RII (CD32), including isoforms Fc.gamma.RIIa
(including allotypes H131 and R131), Fc.gamma.RIIb (including
Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and
Fc.gamma.RIII (CD16), including isoforms Fc.gamma.RIIIa (including
allotypes V158 and F158) and Fc.gamma.RIIIb (including allotypes
Fc.gamma.RIIb-NA1 and Fc.gamma.RIIb-NA2) (Jefferis et al., 2002,
Immunol Lett 82:57-65, entirely incorporated by reference), as well
as any undiscovered human Fc.gamma.Rs or Fc.gamma.R isoforms or
allotypes. An Fc.gamma.R may be from any organism, including but
not limited to humans, mice, rats, rabbits, and monkeys. Mouse
Fc.gamma.Rs include but are not limited to Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and Fc.gamma.RIII-2
(CD16-2), as well as any undiscovered mouse Fc.gamma.Rs or
Fc.gamma.R isoforms or allotypes.
[0171] By "FcRn" or "neonatal Fc Receptor" as used herein is meant
a protein that binds the IgG antibody Fc region and is encoded at
least in part by an FcRn gene. The FcRn may be from any organism,
including but not limited to humans, mice, rats, rabbits, and
monkeys. As is known in the art, the functional FcRn protein
comprises two polypeptides, often referred to as the heavy chain
and light chain. The light chain is beta-2-microglobulin and the
heavy chain is encoded by the FcRn gene. Unless otherwise noted
herein, FcRn or an FcRn protein refers to the complex of FcRn heavy
chain with beta-2-microglobulin. A variety of FcRn variants used to
increase binding to the FcRn receptor, and in some cases, to
increase serum half-life, are shown in the Figure Legend of FIG.
83.
[0172] By "parent polypeptide" as used herein is meant a starting
polypeptide that is subsequently modified to generate a variant.
The parent polypeptide may be a naturally occurring polypeptide, or
a variant or engineered version of a naturally occurring
polypeptide. Parent polypeptide may refer to the polypeptide
itself, compositions that comprise the parent polypeptide, or the
amino acid sequence that encodes it. Accordingly, by "parent
immunoglobulin" as used herein is meant an unmodified
immunoglobulin polypeptide that is modified to generate a variant,
and by "parent antibody" as used herein is meant an unmodified
antibody that is modified to generate a variant antibody. It should
be noted that "parent antibody" includes known commercial,
recombinantly produced antibodies as outlined below.
[0173] By "Fc" or "Fc region" or "Fc domain" as used herein is
meant the polypeptide comprising the constant region of an antibody
excluding the first constant region immunoglobulin domain and in
some cases, part of the hinge. Thus Fc refers to the last two
constant region immunoglobulin domains of IgA, IgD, and IgG, the
last three constant region immunoglobulin domains of IgE and IgM,
and the flexible hinge N-terminal to these domains. For IgA and
IgM, Fc may include the J chain. For IgG, the Fc domain comprises
immunoglobulin domains C.gamma.2 and C.gamma.3 (C.gamma.2 and
C.gamma.3) and the lower hinge region between C.gamma.1 (C.gamma.1)
and C.gamma.2 (C.gamma.2). Although the boundaries of the Fc region
may vary, the human IgG heavy chain Fc region is usually defined to
include residues C226 or P230 to its carboxyl-terminus, wherein the
numbering is according to the EU index as in Kabat. In some
embodiments, as is more fully described below, amino acid
modifications are made to the Fc region, for example to alter
binding to one or more Fc.gamma.R receptors or to the FcRn
receptor.
[0174] By "heavy constant region" herein is meant the
CH1-hinge-CH2-CH3 portion of an antibody.
[0175] By "Fc fusion protein" or "immunoadhesin" herein is meant a
protein comprising an Fc region, generally linked (optionally
through a linker moiety, as described herein) to a different
protein, such as a binding moiety to a target protein, as described
herein. In some cases, one monomer of the heterodimeric antibody
comprises an antibody heavy chain (either including an scFv or
further including a light chain) and the other monomer is a Fc
fusion, comprising a variant Fc domain and a ligand. In some
embodiments, these "half antibody-half fusion proteins" are
referred to as "Fusionbodies".
[0176] By "position" as used herein is meant a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example the EU index for
antibody numbering.
[0177] By "target antigen" as used herein is meant the molecule
that is bound specifically by the variable region of a given
antibody. A target antigen may be a protein, carbohydrate, lipid,
or other chemical compound. Suitable target antigens are described
below.
[0178] By "strandedness" in the context of the monomers of the
heterodimeric antibodies of the invention herein is meant that,
similar to the two strands of DNA that "match", heterodimerization
variants are incorporated into each monomer so as to preserve the
ability to "match" to form heterodimers. For example, if some pI
variants are engineered into monomer A (e.g. making the pI higher)
then steric variants that are "charge pairs" that can be utilized
as well do not interfere with the pI variants, e.g. the charge
variants that make a pI higher are put on the same "strand" or
"monomer" to preserve both functionalities. Similarly, for "skew"
variants that come in pairs of a set as more fully outlined below,
the skilled artisan will consider pI in deciding into which strand
or monomer that incorporates one set of the pair will go, such that
pI separation is maximized using the pI of the skews as well.
[0179] By "target cell" as used herein is meant a cell that
expresses a target antigen.
[0180] By "variable region" as used herein is meant the region of
an immunoglobulin that comprises one or more Ig domains
substantially encoded by any of the V.kappa., V.lamda., and/or VH
genes that make up the kappa, lambda, and heavy chain
immunoglobulin genetic loci respectively.
[0181] By "wild type or WT" herein is meant an amino acid sequence
or a nucleotide sequence that is found in nature, including allelic
variations. A WT protein has an amino acid sequence or a nucleotide
sequence that has not been intentionally modified.
[0182] The antibodies of the present invention are generally
isolated or recombinant. "Isolated," when used to describe the
various polypeptides disclosed herein, means a polypeptide that has
been identified and separated and/or recovered from a cell or cell
culture from which it was expressed. Ordinarily, an isolated
polypeptide will be prepared by at least one purification step. An
"isolated antibody," refers to an antibody which is substantially
free of other antibodies having different antigenic specificities.
"Recombinant" means the antibodies are generated using recombinant
nucleic acid techniques in exogeneous host cells.
[0183] "Percent (%) amino acid sequence identity" with respect to a
protein sequence is defined as the percentage of amino acid
residues in a candidate sequence that are identical with the amino
acid residues in the specific (parental) sequence, after aligning
the sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity, and not considering any
conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. One particular program is the ALIGN-2 program
outlined at paragraphs [0279] to [0280] of US Pub. No. 20160244525,
hereby incorporated by reference.
[0184] The degree of identity between an amino acid sequence of the
present invention ("invention sequence") and the parental amino
acid sequence is calculated as the number of exact matches in an
alignment of the two sequences, divided by the length of the
"invention sequence," or the length of the parental sequence,
whichever is the shortest. The result is expressed in percent
identity.
[0185] In some embodiments, two or more amino acid sequences are at
least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments,
two or more amino acid sequences are at least 95%, 97%, 98%, 99%,
or even 100% identical.
[0186] "Specific binding" or "specifically binds to" or is
"specific for" a particular antigen or an epitope means binding
that is measurably different from a non-specific interaction.
Specific binding can be measured, for example, by determining
binding of a molecule compared to binding of a control molecule,
which generally is a molecule of similar structure that does not
have binding activity. For example, specific binding can be
determined by competition with a control molecule that is similar
to the target.
[0187] Specific binding for a particular antigen or an epitope can
be exhibited, for example, by an antibody having a KD for an
antigen or epitope of at least about 10.sup.-4 M, at least about
10.sup.-5M, at least about 10.sup.-6 M, at least about 10.sup.-7 M,
at least about 10.sup.-8 M, at least about 10.sup.-9 M,
alternatively at least about 10.sup.-10 M, at least about
10.sup.-11 M, at least about 10.sup.-12 M, or greater, where KD
refers to a dissociation rate of a particular antibody-antigen
interaction. Typically, an antibody that specifically binds an
antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-,
10,000- or more times greater for a control molecule relative to
the antigen or epitope.
[0188] Also, specific binding for a particular antigen or an
epitope can be exhibited, for example, by an antibody having a KA
or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-,
1000-, 5,000-, 10,000- or more times greater for the epitope
relative to a control, where KA or Ka refers to an association rate
of a particular antibody-antigen interaction. Binding affinity is
generally measured using a BIACORE.RTM. assay.
V. Antibodies
[0189] The present invention relates to the generation of
bispecific immunomodulatory antibodies that bind two different
immunomodulatory antigens as discussed herein. As is discussed
below, the term "antibody" is used generally. Antibodies that find
use in the present invention can take on a number of formats as
described herein.
[0190] Traditional antibody structural units typically comprise a
tetramer. Each tetramer is typically composed of two identical
pairs of polypeptide chains, each pair having one "light"
(typically having a molecular weight of about 25 kDa) and one
"heavy" chain (typically having a molecular weight of about 50-70
kDa). Human light chains are classified as kappa and lambda light
chains. The present invention is directed to bispecific antibodies
that generally are based on the IgG class, which has several
subclasses, including, but not limited to IgG1, IgG2, IgG3, and
IgG4. In general, IgG1, IgG2 and IgG4 are used more frequently than
IgG3. It should be noted that IgG1 has different allotypes with
polymorphisms at 356 (D or E) and 358 (L or M). The sequences
depicted herein use the 356D/358M allotype, however the other
allotype is included herein. That is, any sequence inclusive of an
IgG1 Fc domain included herein can have 356E/358L replacing the
356D/358M allotype.
[0191] In addition, many of the sequences herein have at least one
of the cysteines at position 220 replaced by a serine; generally,
this is the on the "scFv monomer" side for most of the sequences
depicted herein, although it can also be on the "Fab monomer" side,
or both, to reduce disulfide formation. Specifically included
within the sequences herein are one or both of these cysteines
replaced (C220S).
[0192] Thus, "isotype" as used herein is meant any of the
subclasses of immunoglobulins defined by the chemical and antigenic
characteristics of their constant regions. It should be understood
that therapeutic antibodies can also comprise hybrids of isotypes
and/or subclasses. For example, as shown in US Publication
2009/0163699, incorporated by reference, the present invention
covers pI engineering of IgG1/G2 hybrids.
[0193] The amino-terminal portion of each chain includes a variable
region of about 100 to 110 or more amino acids primarily
responsible for antigen recognition, generally referred to in the
art and herein as the "Fv domain" or "Fv region". In the variable
region, three loops are gathered for each of the V domains of the
heavy chain and light chain to form an antigen-binding site. Each
of the loops is referred to as a complementarity-determining region
(hereinafter referred to as a "CDR"), in which the variation in the
amino acid sequence is most significant. "Variable" refers to the
fact that certain segments of the variable region differ
extensively in sequence among antibodies. Variability within the
variable region is not evenly distributed. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-15 amino
acids long or longer.
[0194] Each VH and VL is composed of three hypervariable regions
("complementary determining regions," "CDRs") and four FRs,
arranged from amino-terminus to carboxy-terminus in the following
order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
[0195] The hypervariable region generally encompasses amino acid
residues from about amino acid residues 24-34 (LCDR1; "L" denotes
light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain
variable region and around about 31-35B (HCDR1; "H" denotes heavy
chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain
variable region; Kabat et al., SEQUENCES OF PROTEINS OF
IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991) and/or those residues
forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52
(LCDR2) and 91-96 (LCDR3) in the light chain variable region and
26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain
variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.
Specific CDRs of the invention are described below.
[0196] As will be appreciated by those in the art, the exact
numbering and placement of the CDRs can be different among
different numbering systems. However, it should be understood that
the disclosure of a variable heavy and/or variable light sequence
includes the disclosure of the associated (inherent) CDRs.
Accordingly, the disclosure of each variable heavy region is a
disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2 and vhCDR3) and the
disclosure of each variable light region is a disclosure of the
vlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3).
[0197] A useful comparison of CDR numbering is as below, see
Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003):
TABLE-US-00001 TABLE 1 Kabat + Chothia IMGT Kabat AbM Chothia
Contact Xencor vhCDR1 26-35 27-38 31-35 26-35 26-32 30-35 27-35
vhCDR2 50-65 56-65 50-65 50-58 52-56 47-58 54-61 vhCDR3 95-102
105-117 95-102 95-102 95-102 93-101 103-116 vlCDR1 24-34 27-38
24-34 24-34 24-34 30-36 27-38 vlCDR2 50-56 56-65 50-56 50-56 50-56
46-55 56-62 vlCDR3 89-97 105-117 89-97 89-97 89-97 89-96 97-105
[0198] Throughout the present specification, the Kabat numbering
system is generally used when referring to a residue in the
variable domain (approximately, residues 1-107 of the light chain
variable region and residues 1-113 of the heavy chain variable
region) and the EU numbering system for Fc regions (e.g, Kabat et
al., supra (1991)).
[0199] The present invention provides a large number of different
CDR sets. In this case, a "full CDR set" comprises the three
variable light and three variable heavy CDRs, e.g. a vlCDR1,
vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. These can be part of a
larger variable light or variable heavy domain, respectfully. In
addition, as more fully outlined herein, the variable heavy and
variable light domains can be on separate polypeptide chains, when
a heavy and light chain is used (for example when Fabs are used),
or on a single polypeptide chain in the case of scFv sequences.
[0200] The CDRs contribute to the formation of the antigen-binding,
or more specifically, epitope binding site of antibodies. "Epitope"
refers to a determinant that interacts with a specific antigen
binding site in the variable region of an antibody molecule known
as a paratope. Epitopes are groupings of molecules such as amino
acids or sugar side chains and usually have specific structural
characteristics, as well as specific charge characteristics. A
single antigen may have more than one epitope.
[0201] The epitope may comprise amino acid residues directly
involved in the binding (also called immunodominant component of
the epitope) and other amino acid residues, which are not directly
involved in the binding, such as amino acid residues which are
effectively blocked by the specifically antigen binding peptide; in
other words, the amino acid residue is within the footprint of the
specifically antigen binding peptide.
[0202] Epitopes may be either conformational or linear. A
conformational epitope is produced by spatially juxtaposed amino
acids from different segments of the linear polypeptide chain. A
linear epitope is one produced by adjacent amino acid residues in a
polypeptide chain. Conformational and nonconformational epitopes
may be distinguished in that the binding to the former but not the
latter is lost in the presence of denaturing solvents.
[0203] An epitope typically includes at least 3, and more usually,
at least 5 or 8-10 amino acids in a unique spatial conformation.
Antibodies that recognize the same epitope can be verified in a
simple immunoassay showing the ability of one antibody to block the
binding of another antibody to a target antigen, for example
"binning." As outlined below, the invention not only includes the
enumerated antigen binding domains and antibodies herein, but those
that compete for binding with the epitopes bound by the enumerated
antigen binding domains.
[0204] The carboxy-terminal portion of each chain defines a
constant region primarily responsible for effector function. Kabat
et al. collected numerous primary sequences of the variable regions
of heavy chains and light chains. Based on the degree of
conservation of the sequences, they classified individual primary
sequences into the CDR and the framework and made a list thereof
(see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH
publication, No. 91-3242, E. A. Kabat et al., entirely incorporated
by reference).
[0205] In the IgG subclass of immunoglobulins, there are several
immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig)
domain" herein is meant a region of an immunoglobulin having a
distinct tertiary structure. Of interest in the present invention
are the heavy chain domains, including, the constant heavy (CH)
domains and the hinge domains. In the context of IgG antibodies,
the IgG isotypes each have three CH regions. Accordingly, "CH"
domains in the context of IgG are as follows: "CH1" refers to
positions 118-220 according to the EU index as in Kabat. "CH2"
refers to positions 237-340 according to the EU index as in Kabat,
and "CH3" refers to positions 341-447 according to the EU index as
in Kabat. As shown herein and described below, the pI variants can
be in one or more of the CH regions, as well as the hinge region,
discussed below.
[0206] Another type of Ig domain of the heavy chain is the hinge
region. By "hinge" or "hinge region" or "antibody hinge region" or
"immunoglobulin hinge region" herein is meant the flexible
polypeptide comprising the amino acids between the first and second
constant domains of an antibody. Structurally, the IgG CH1 domain
ends at EU position 220, and the IgG CH2 domain begins at residue
EU position 237. Thus for IgG the antibody hinge is herein defined
to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1),
wherein the numbering is according to the EU index as in Kabat. In
some embodiments, for example in the context of an Fc region, the
lower hinge is included, with the "lower hinge" generally referring
to positions 226 or 230. As noted herein, pI variants can be made
in the hinge region as well.
[0207] The light chain generally comprises two domains, the
variable light domain (containing the light chain CDRs and together
with the variable heavy domains forming the Fv region), and a
constant light chain region (often referred to as CL or
C.kappa.).
[0208] Another region of interest for additional substitutions,
outlined below, is the Fc region.
[0209] Thus, the present invention provides different antibody
domains. As described herein and known in the art, the
heterodimeric antibodies of the invention comprise different
domains within the heavy and light chains, which can be overlapping
as well. These domains include, but are not limited to, the Fc
domain, the CH1 domain, the CH2 domain, the CH3 domain, the hinge
domain, the heavy constant domain (CH1-hinge-Fc domain or
CH1-hinge-CH2-CH3), the variable heavy domain, the variable light
domain, the light constant domain, Fab domains and scFv
domains.
[0210] Thus, the "Fc domain" includes the --CH2-CH3 domain, and
optionally a hinge domain. In the embodiments herein, when a scFv
is attached to an Fc domain, it is the C-terminus of the scFv
construct that is attached to all or part of the hinge of the Fc
domain; for example, it is generally attached to the sequence EPKS
which is the beginning of the hinge. The heavy chain comprises a
variable heavy domain and a constant domain, which includes a
CH1-optional hinge-Fc domain comprising a CH2-CH3. The light chain
comprises a variable light chain and the light constant domain. A
scFv comprises a variable heavy chain, an scFv linker, and a
variable light domain. In most of the constructs and sequences
outlined herein, C-terminus of the variable light chain is attached
to the N-terminus of the scFv linker, the C-terminus of which is
attached to the N-terminus of a variable heavy chain
(N-vh-linker-vl-C) although that can be switched
(N-vl-linker-vh-C). Some embodiments of the invention comprise at
least one scFv domain, which, while not naturally occurring,
generally includes a variable heavy domain and a variable light
domain, linked together by a scFv linker. As outlined herein, while
the scFv domain is generally from N- to C-terminus oriented as
vh-scFv linker-vl, this can be reversed for any of the scFv domains
(or those constructed using vh and vl sequences from Fabs), to
vl-scFv linker-vh, with optional linkers at one or both ends
depending on the format (see generally FIG. 2).
[0211] As shown herein, there are a number of suitable scFv linkers
that can be used, including traditional peptide bonds, generated by
recombinant techniques. The linker peptide may predominantly
include the following amino acid residues: Gly, Ser, Ala, or Thr.
The linker peptide should have a length that is adequate to link
two molecules in such a way that they assume the correct
conformation relative to one another so that they retain the
desired activity. In one embodiment, the linker is from about 1 to
50 amino acids in length, preferably about 1 to 30 amino acids in
length. In one embodiment, linkers of 1 to 20 amino acids in length
may be used, with from about 5 to about 10 amino acids finding use
in some embodiments. Useful linkers include glycine-serine
polymers, including for example (GS)n, (GSGGS)n, (GGGGS)n, and
(GGGS)n, where n is an integer of at least one (and generally from
3 to 4), glycine-alanine polymers, alanine-serine polymers, and
other flexible linkers. Alternatively, a variety of
nonproteinaceous polymers, including but not limited to
polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes,
or copolymers of polyethylene glycol and polypropylene glycol, may
find use as linkers, that is may find use as linkers.
[0212] Other linker sequences may include any sequence of any
length of CL/CH1 domain but not all residues of CL/CH1 domain; for
example the first 5-12 amino acid residues of the CL/CH1 domains.
Linkers can be derived from immunoglobulin light chain, for example
C.kappa. or C.lamda.. Linkers can be derived from immunoglobulin
heavy chains of any isotype, including for example C.gamma.1,
C.gamma.2, C.gamma.3, C.gamma.4, C.alpha.1, C.alpha.2, C.delta.,
C.epsilon., and C.mu.. Linker sequences may also be derived from
other proteins such as Ig-like proteins (e.g. TCR, FcR, KIR), hinge
region-derived sequences, and other natural sequences from other
proteins.
[0213] In some embodiments, the linker is a "domain linker", used
to link any two domains as outlined herein together. While any
suitable linker can be used, many embodiments utilize a
glycine-serine polymer, including for example (GS)n, (GSGGS)n,
(GGGGS)n, and (GGGS)n, where n is an integer of at least one (and
generally from 3 to 4 to 5) as well as any peptide sequence that
allows for recombinant attachment of the two domains with
sufficient length and flexibility to allow each domain to retain
its biological function. In some cases, and with attention being
paid to "strandedness", as outlined below, charged domain linkers,
as used in some embodiments of scFv linkers can be used.
[0214] In some embodiments, the scFv linker is a charged scFv
linker, a number of which are shown in FIG. 8. Accordingly, the
present invention further provides charged scFv linkers, to
facilitate the separation in pI between a first and a second
monomer. That is, by incorporating a charged scFv linker, either
positive or negative (or both, in the case of scaffolds that use
scFvs on different monomers), this allows the monomer comprising
the charged linker to alter the pI without making further changes
in the Fc domains. These charged linkers can be substituted into
any scFv containing standard linkers. Again, as will be appreciated
by those in the art, charged scFv linkers are used on the correct
"strand" or monomer, according to the desired changes in pI. For
example, as discussed herein, to make triple F format heterodimeric
antibody, the original pI of the Fv region for each of the desired
antigen binding domains are calculated, and one is chosen to make
an scFv, and depending on the pI, either positive or negative
linkers are chosen.
[0215] Charged domain linkers can also be used to increase the pI
separation of the monomers of the invention as well, and thus those
included in FIG. 8 can be used in any embodiment herein where a
linker is utilized.
[0216] In one embodiment, the antibody is an antibody fragment, as
long as it contains at least one constant domain which can be
engineered to produce heterodimers, such as pI engineering. Other
antibody fragments that can be used include fragments that contain
one or more of the CH1, CH2, CH3, hinge and CL domains of the
invention that have been pI engineered. In particular, the formats
depicted in FIG. 1 are antibodies, usually referred to as
"heterodimeric antibodies", meaning that the protein has at least
two associated Fc sequences self-assembled into a heterodimeric Fc
domain and at least two Fv regions, whether as Fabs or as
scFvs.
A. Chimeric and Humanized Antibodies
[0217] In some embodiments, the antibodies herein can be derived
from a mixture from different species, e.g. a chimeric antibody
and/or a humanized antibody. In general, both "chimeric antibodies"
and "humanized antibodies" refer to antibodies that combine regions
from more than one species. For example, "chimeric antibodies"
traditionally comprise variable region(s) from a mouse (or rat, in
some cases) and the constant region(s) from a human. "Humanized
antibodies" generally refer to non-human antibodies that have had
the variable-domain framework regions swapped for sequences found
in human antibodies. Generally, in a humanized antibody, the entire
antibody, except the CDRs, is encoded by a polynucleotide of human
origin or is identical to such an antibody except within its CDRs.
The CDRs, some or all of which are encoded by nucleic acids
originating in a non-human organism, are grafted into the
beta-sheet framework of a human antibody variable region to create
an antibody, the specificity of which is determined by the
engrafted CDRs. The creation of such antibodies is described in,
e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et
al., 1988, Science 239:1534-1536, all entirely incorporated by
reference. "Backmutation" of selected acceptor framework residues
to the corresponding donor residues is often required to regain
affinity that is lost in the initial grafted construct (U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370;
5,859,205; 5,821,337; 6,054,297; 6,407,213, all entirely
incorporated by reference). The humanized antibody optimally also
will comprise at least a portion of an immunoglobulin constant
region, typically that of a human immunoglobulin, and thus will
typically comprise a human Fc region. Humanized antibodies can also
be generated using mice with a genetically engineered immune
system. Roque et al., 2004, Biotechnol. Prog. 20:639-654, entirely
incorporated by reference. A variety of techniques and methods for
humanizing and reshaping non-human antibodies are well known in the
art (See Tsurushita & Vasquez, 2004, Humanization of Monoclonal
Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science
(USA), and references cited therein, all entirely incorporated by
reference). Humanization methods include but are not limited to
methods described in Jones et al., 1986, Nature 321:522-525;
Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988,
Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA
86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et
al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997,
Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad.
Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8,
all entirely incorporated by reference. Humanization or other
methods of reducing the immunogenicity of nonhuman antibody
variable regions may include resurfacing methods, as described for
example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA
91:969-973, entirely incorporated by reference.
[0218] In certain embodiments, the antibodies of the invention
comprise a heavy chain variable region from a particular germline
heavy chain immunoglobulin gene and/or a light chain variable
region from a particular germline light chain immunoglobulin gene.
For example, such antibodies may comprise or consist of a human
antibody comprising heavy or light chain variable regions that are
"the product of" or "derived from" a particular germline sequence.
A human antibody that is "the product of" or "derived from" a human
germline immunoglobulin sequence can be identified as such by
comparing the amino acid sequence of the human antibody to the
amino acid sequences of human germline immunoglobulins and
selecting the human germline immunoglobulin sequence that is
closest in sequence (i.e., greatest % identity) to the sequence of
the human antibody. A human antibody that is "the product of" or
"derived from" a particular human germline immunoglobulin sequence
may contain amino acid differences as compared to the germline
sequence, due to, for example, naturally-occurring somatic
mutations or intentional introduction of site-directed mutation.
However, a humanized antibody typically is at least 90% identical
in amino acids sequence to an amino acid sequence encoded by a
human germline immunoglobulin gene and contains amino acid residues
that identify the antibody as being derived from human sequences
when compared to the germline immunoglobulin amino acid sequences
of other species (e.g., murine germline sequences). In certain
cases, a humanized antibody may be at least 95, 96, 97, 98 or 99%,
or even at least 96%, 97%, 98%, or 99% identical in amino acid
sequence to the amino acid sequence encoded by the germline
immunoglobulin gene. Typically, a humanized antibody derived from a
particular human germline sequence will display no more than 10-20
amino acid differences from the amino acid sequence encoded by the
human germline immunoglobulin gene (prior to the introduction of
any skew, pI and ablation variants herein; that is, the number of
variants is generally low, prior to the introduction of the
variants of the invention). In certain cases, the humanized
antibody may display no more than 5, or even no more than 4, 3, 2,
or 1 amino acid difference from the amino acid sequence encoded by
the germline immunoglobulin gene (again, prior to the introduction
of any skew, pI and ablation variants herein; that is, the number
of variants is generally low, prior to the introduction of the
variants of the invention).
[0219] In one embodiment, the parent antibody has been affinity
matured, as is known in the art. Structure-based methods may be
employed for humanization and affinity maturation, for example as
described in U.S. Ser. No. 11/004,590. Selection based methods may
be employed to humanize and/or affinity mature antibody variable
regions, including but not limited to methods described in Wu et
al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol.
Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem.
271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.
USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering
16(10):753-759, all entirely incorporated by reference. Other
humanization methods may involve the grafting of only parts of the
CDRs, including but not limited to methods described in U.S. Ser.
No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De
Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirely
incorporated by reference.
VI. Heterodimeric Antibodies
[0220] Accordingly, in some embodiments the present invention
provides heterodimeric immunomodulatory antibodies that rely on the
use of two different heavy chain variant Fc sequences, that will
self-assemble to form heterodimeric Fc domains and heterodimeric
antibodies.
[0221] The present invention is directed to novel constructs to
provide heterodimeric antibodies that allow binding to more than
one immunomodulatory antigen or ligand, e.g. to allow for
bispecific binding. The heterodimeric antibody constructs are based
on the self-assembling nature of the two Fc domains of the heavy
chains of antibodies, e.g. two "monomers" that assemble into a
"dimer". Heterodimeric antibodies are made by altering the amino
acid sequence of each monomer as more fully discussed below. Thus,
the present invention is generally directed to the creation of
heterodimeric immunomodulatory antibodies which can co-engage
antigens in several ways, relying on amino acid variants in the
constant regions that are different on each chain to promote
heterodimeric formation and/or allow for ease of purification of
heterodimers over the homodimers.
[0222] Thus, the present invention provides bispecific antibodies.
An ongoing problem in antibody technologies is the desire for
"bispecific" antibodies that bind to two different antigens
simultaneously, in general thus allowing the different antigens to
be brought into proximity and resulting in new functionalities and
new therapies. In general, these antibodies are made by including
genes for each heavy and light chain into the host cells. This
generally results in the formation of the desired heterodimer
(A-B), as well as the two homodimers (A-A and B-B (not including
the light chain heterodimeric issues)). However, a major obstacle
in the formation of bispecific antibodies is the difficulty in
purifying the heterodimeric antibodies away from the homodimeric
antibodies and/or biasing the formation of the heterodimer over the
formation of the homodimers.
[0223] There are a number of mechanisms that can be used to
generate the heterodimers of the present invention. In addition, as
will be appreciated by those in the art, these mechanisms can be
combined to ensure high heterodimerization. Thus, amino acid
variants that lead to the production of heterodimers are referred
to as "heterodimerization variants". As discussed below,
heterodimerization variants can include steric variants (e.g. the
"knobs and holes" or "skew" variants described below and the
"charge pairs" variants described below) as well as "pI variants",
which allows purification of homodimers away from heterodimers. As
is generally described in WO2014/145806, hereby incorporated by
reference in its entirety and specifically as below for the
discussion of "heterodimerization variants", useful mechanisms for
heterodimerization include "knobs and holes" ("KIH"; sometimes
herein as "skew" variants (see discussion in WO2014/145806),
"electrostatic steering" or "charge pairs" as described in
WO2014/145806, pI variants as described in WO2014/145806, and
general additional Fc variants as outlined in WO2014/145806 and
below.
[0224] In the present invention, there are several basic mechanisms
that can lead to ease of purifying heterodimeric antibodies; one
relies on the use of pI variants, such that each monomer has a
different pI, thus allowing the isoelectric purification of A-A,
A-B and B-B dimeric proteins. Alternatively, some scaffold formats,
such as the "triple F" format, also allows separation on the basis
of size. As is further outlined below, it is also possible to
"skew" the formation of heterodimers over homodimers. Thus, a
combination of steric heterodimerization variants and pI or charge
pair variants find particular use in the invention.
[0225] In general, embodiments of particular use in the present
invention rely on sets of variants that include skew variants, that
encourage heterodimerization formation over homodimerization
formation, coupled with pI variants, which increase the pI
difference between the two monomers.
[0226] Additionally, as more fully outlined below, depending on the
format of the heterodimer antibody, pI variants can be either
contained within the constant and/or Fc domains of a monomer, or
charged linkers, either domain linkers or scFv linkers, can be
used. That is, scaffolds that utilize scFv(s) such as the Triple F
format can include charged scFv linkers (either positive or
negative), that give a further pI boost for purification purposes.
As will be appreciated by those in the art, some Triple F formats
are useful with just charged scFv linkers and no additional pI
adjustments, although the invention does provide pI variants that
are on one or both of the monomers, and/or charged domain linkers
as well. In addition, additional amino acid engineering for
alternative functionalities may also confer pI changes, such as Fc,
FcRn and KO variants.
[0227] In the present invention that utilizes pI as a separation
mechanism to allow the purification of heterodimeric proteins,
amino acid variants can be introduced into one or both of the
monomer polypeptides; that is, the pI of one of the monomers
(referred to herein for simplicity as "monomer A") can be
engineered away from monomer B, or both monomer A and B change be
changed, with the pI of monomer A increasing and the pI of monomer
B decreasing. As discussed, the pI changes of either or both
monomers can be done by removing or adding a charged residue (e.g.
a neutral amino acid is replaced by a positively or negatively
charged amino acid residue, e.g. glycine to glutamic acid),
changing a charged residue from positive or negative to the
opposite charge (e.g. aspartic acid to lysine) or changing a
charged residue to a neutral residue (e.g. loss of a charge; lysine
to serine). A number of these variants are shown in the
Figures.
[0228] Accordingly, this embodiment of the present invention
provides for creating a sufficient change in pI in at least one of
the monomers such that heterodimers can be separated from
homodimers. As will be appreciated by those in the art, and as
discussed further below, this can be done by using a "wild type"
heavy chain constant region and a variant region that has been
engineered to either increase or decrease its pI (wt A-+B or wt
A--B), or by increasing one region and decreasing the other region
(A+-B- or A-B+).
[0229] Thus, in general, a component of some embodiments of the
present invention are amino acid variants in the constant regions
of antibodies that are directed to altering the isoelectric point
(pI) of at least one, if not both, of the monomers of a dimeric
protein to form "pI antibodies" by incorporating amino acid
substitutions ("pI variants" or "pI substitutions") into one or
both of the monomers. As shown herein, the separation of the
heterodimers from the two homodimers can be accomplished if the pIs
of the two monomers differ by as little as 0.1 pH unit, with 0.2,
0.3, 0.4 and 0.5 or greater all finding use in the present
invention. As will be appreciated by those in the art, the number
of pI variants to be included on each or both monomer(s) to get
good separation will depend in part on the starting pI of the
components, for example in the triple F format, the starting pI of
the scFv and Fab of interest. That is, to determine which monomer
to engineer or in which "direction" (e.g. more positive or more
negative), the Fv sequences of the two target antigens are
calculated and a decision is made from there. As is known in the
art, different Fvs will have different starting pIs which are
exploited in the present invention. In general, as outlined herein,
the pIs are engineered to result in a total pI difference of each
monomer of at least about 0.1 logs, with 0.2 to 0.5 being preferred
as outlined herein. Furthermore, as will be appreciated by those in
the art and outlined herein, in some embodiments, heterodimers can
be separated from homodimers on the basis of size. As shown in FIG.
2, for example, several of the formats allow separation of
heterodimers and homodimers on the basis of size.
A. Heterodimerization Variants
[0230] The present invention provides heterodimeric proteins,
including heterodimeric antibodies in a variety of formats, which
utilize heterodimeric variants to allow for heterodimeric formation
and/or purification away from homodimers. A number of
heterodimerization variants are shown in FIG. 4.
[0231] There are a number of suitable pairs of sets of
heterodimerization skew variants. These variants come in "pairs" of
"sets". That is, one set of the pair is incorporated into the first
monomer and the other set of the pair is incorporated into the
second monomer. It should be noted that these sets do not
necessarily behave as "knobs in holes" variants, with a one-to-one
correspondence between a residue on one monomer and a residue on
the other; that is, these pairs of sets form an interface between
the two monomers that encourages heterodimer formation and
discourages homodimer formation, allowing the percentage of
heterodimers that spontaneously form under biological conditions to
be over 90%, rather than the expected 50% (25% homodimer A/A:50%
heterodimer A/B:25% homodimer B/B).
B. Steric Variants
[0232] In some embodiments, the formation of heterodimers can be
facilitated by the addition of steric variants. That is, by
changing amino acids in each heavy chain, different heavy chains
are more likely to associate to form the heterodimeric structure
than to form homodimers with the same Fc amino acid sequences.
Suitable steric variants are included in in the Figures.
[0233] One mechanism is generally referred to in the art as "knobs
and holes", referring to amino acid engineering that creates steric
influences to favor heterodimeric formation and disfavor
homodimeric formation can also optionally be used; this is
sometimes referred to as "knobs and holes", as described in U.S.
Ser. No. 61/596,846, Ridgway et al., Protein Engineering 9(7):617
(1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S. Pat. No.
8,216,805, all of which are hereby incorporated by reference in
their entirety. The Figures identify a number of "monomer A-monomer
B" pairs that rely on "knobs and holes". In addition, as described
in Merchant et al., Nature Biotech. 16:677 (1998), these "knobs and
hole" mutations can be combined with disulfide bonds to skew
formation to heterodimerization.
[0234] An additional mechanism that finds use in the generation of
heterodimers is sometimes referred to as "electrostatic steering"
as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637
(2010), hereby incorporated by reference in its entirety. This is
sometimes referred to herein as "charge pairs". In this embodiment,
electrostatics are used to skew the formation towards
heterodimerization. As those in the art will appreciate, these may
also have have an effect on pI, and thus on purification, and thus
could in some cases also be considered pI variants. However, as
these were generated to force heterodimerization and were not used
as purification tools, they are classified as "steric variants".
These include, but are not limited to, D221E/P228E/L368E paired
with D221R/P228R/K409R (e.g. these are "monomer corresponding sets)
and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.
[0235] Additional monomer A and monomer B variants that can be
combined with other variants, optionally and independently in any
amount, such as pI variants outlined herein or other steric
variants that are shown in FIG. 37 of US 2012/0149876, the figure
and legend and SEQ ID NOs of which are incorporated expressly by
reference herein.
[0236] In some embodiments, the steric variants outlined herein can
be optionally and independently incorporated with any pI variant
(or other variants such as Fc variants, FcRn variants, etc.) into
one or both monomers, and can be independently and optionally
included or excluded from the proteins of the invention.
[0237] A list of suitable skew variants is found in FIG. 4 showing
some pairs of particular utility in many embodiments. Of particular
use in many embodiments are the pairs of sets including, but not
limited to, S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K;
L368D/K370S:S364K/E357L, K370S:S364K/E357Q and
T366S/L368A/Y407V:T366W (optionally including a bridging disulfide,
T366S/L368A/Y407V/Y349C:T366W/S354C). In terms of nomenclature, the
pair "S364K/E357Q:L368D/K370S" means that one of the monomers has
the double variant set S364K/E357Q and the other has the double
variant set L368D/K370S; as above, the "strandedness" of these
pairs depends on the starting pI.
C. pI (Isoelectric Point) Variants for Heterodimers
[0238] In general, as will be appreciated by those in the art,
there are two general categories of pI variants: those that
increase the pI of the protein (basic changes) and those that
decrease the pI of the protein (acidic changes). As described
herein, all combinations of these variants can be done: one monomer
may be wild type, or a variant that does not display a
significantly different pI from wild-type, and the other can be
either more basic or more acidic. Alternatively, each monomer is
changed, one to more basic and one to more acidic.
[0239] Preferred combinations of pI variants are shown in FIG. 5.
As outlined herein and shown in the figures, these changes are
shown relative to IgG1, but all isotypes can be altered this way,
as well as isotype hybrids. In the case where the heavy chain
constant domain is from IgG2-4, R133E and R133Q can also be
used.
[0240] In one embodiment, for example in the bottle opener format,
a preferred combination of pI variants has one monomer (the
negative Fab side) comprising 208D/295E/384D/418E/421D variants
(N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) and a
second monomer (the positive scFv side) comprising a positively
charged scFv linker, including (GKPGS).sub.4. However, as will be
appreciated by those in the art, the first monomer includes a CH1
domain, including position 208. Accordingly, in constructs that do
not include a CH1 domain (for example for heterodimeric Fc fusion
proteins that do not utilize a CH1 domain on one of the domains,
for example in a dual scFv format), a preferred negative pI variant
Fc set includes 295E/384D/418E/421D variants
(Q295E/N384D/Q418E/N421D when relative to human IgG1).
[0241] Accordingly, in some embodiments, one monomer has a set of
substitutions from FIG. 5 and the other monomer has a charged
linker (either in the form of a charged scFv linker because that
monomer comprises an scFv or a charged domain linker, as the format
dictates).
[0242] 1. Isotypic Variants
[0243] In addition, many embodiments of the invention rely on the
"importation" of pI amino acids at particular positions from one
IgG isotype into another, thus reducing or eliminating the
possibility of unwanted immunogenicity being introduced into the
variants. A number of these are shown in FIG. 21 of US Publ.
2014/0370013, hereby incorporated by reference. That is, IgG1 is a
common isotype for therapeutic antibodies for a variety of reasons,
including high effector function. However, the heavy constant
region of IgG1 has a higher pI than that of IgG2 (8.10 versus
7.31). By introducing IgG2 residues at particular positions into
the IgG1 backbone, the pI of the resulting monomer is lowered (or
increased) and additionally exhibits longer serum half-life. For
example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 has
a glutamic acid (pI 3.22); importing the glutamic acid will affect
the pI of the resulting protein. As is described below, a number of
amino acid substitutions are generally required to significant
affect the pI of the variant antibody. However, it should be noted
as discussed below that even changes in IgG2 molecules allow for
increased serum half-life.
[0244] In other embodiments, non-isotypic amino acid changes are
made, either to reduce the overall charge state of the resulting
protein (e.g. by changing a higher pI amino acid to a lower pI
amino acid), or to allow accommodations in structure for stability,
etc. as is more further described below.
[0245] In addition, by pI engineering both the heavy and light
constant domains, significant changes in each monomer of the
heterodimer can be seen. As discussed herein, having the pIs of the
two monomers differ by at least 0.5 can allow separation by ion
exchange chromatography or isoelectric focusing, or other methods
sensitive to isoelectric point.
D. Calculating pI
[0246] The pI of each monomer can depend on the pI of the variant
heavy chain constant domain and the pI of the total monomer,
including the variant heavy chain constant domain and the fusion
partner. Thus, in some embodiments, the change in pI is calculated
on the basis of the variant heavy chain constant domain, using the
chart in the FIG. 19 of US Pub. 2014/0370013. As discussed herein,
which monomer to engineer is generally decided by the inherent pI
of the FAT and scaffold regions. Alternatively, the pI of each
monomer can be compared.
E. pI Variants that Also Confer Better FcRn In Vivo Binding
[0247] In the case where the pI variant decreases the pI of the
monomer, they can have the added benefit of improving serum
retention in vivo.
[0248] Although still under examination, Fc regions are believed to
have longer half-lives in vivo, because binding to FcRn at pH 6 in
an endosome sequesters the Fc (Ghetie and Ward, 1997 Immunol Today.
18(12): 592-598, entirely incorporated by reference). The endosomal
compartment then recycles the Fc to the cell surface. Once the
compartment opens to the extracellular space, the higher pH,
.about.7.4, induces the release of Fc back into the blood. In mice,
Dall'Acqua et al. showed that Fc mutants with increased FcRn
binding at pH 6 and pH 7.4 actually had reduced serum
concentrations and the same half life as wild-type Fc (Dall'Acqua
et al. 2002, J. Immunol. 169:5171-5180, entirely incorporated by
reference). The increased affinity of Fc for FcRn at pH 7.4 is
thought to forbid the release of the Fc back into the blood.
Therefore, the Fc mutations that will increase Fc's half-life in
vivo will ideally increase FcRn binding at the lower pH while still
allowing release of Fc at higher pH. The amino acid histidine
changes its charge state in the pH range of 6.0 to 7.4. Therefore,
it is not surprising to find His residues at important positions in
the Fc/FcRn complex.
[0249] Recently it has been suggested that antibodies with variable
regions that have lower isoelectric points may also have longer
serum half-lives (Igawa et al., 2010 PEDS. 23(5): 385-392, entirely
incorporated by reference). However, the mechanism of this is still
poorly understood. Moreover, variable regions differ from antibody
to antibody. Constant region variants with reduced pI and extended
half-life would provide a more modular approach to improving the
pharmacokinetic properties of antibodies, as described herein.
F. Additional Fc Variants for Additional Functionality
[0250] In addition to pI amino acid variants, there are a number of
useful Fc amino acid modification that can be made for a variety of
reasons, including, but not limited to, altering binding to one or
more Fc.gamma.R receptors, altered binding to FcRn receptors,
etc.
[0251] Accordingly, the proteins of the invention can include amino
acid modifications, including the heterodimerization variants
outlined herein, which includes the pI variants and steric
variants. Each set of variants can be independently and optionally
included or excluded from any particular heterodimeric protein.
G. Fc.gamma.R Variants
[0252] Accordingly, there are a number of useful Fc substitutions
that can be made to alter binding to one or more of the Fc.gamma.R
receptors. Substitutions that result in increased binding as well
as decreased binding can be useful. For example, it is known that
increased binding to Fc.gamma.RIIIa results in increased ADCC
(antibody dependent cell-mediated cytotoxicity; the cell-mediated
reaction wherein nonspecific cytotoxic cells that express
Fc.gamma.Rs recognize bound antibody on a target cell and
subsequently cause lysis of the target cell). Similarly, decreased
binding to Fc.gamma.RIIb (an inhibitory receptor) can be beneficial
as well in some circumstances. Amino acid substitutions that find
use in the present invention include those listed in U.S. Ser. No.
11/124,620 (particularly FIG. 41), Ser. Nos. 11/174,287,
11/396,495, 11/538,406, all of which are expressly incorporated
herein by reference in their entirety and specifically for the
variants disclosed therein. Particular variants that find use
include, but are not limited to, 236A, 239D, 239E, 332E, 332D,
239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y,
239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.
[0253] In addition, there are additional Fc substitutions that find
use in increased binding to the FcRn receptor and increased serum
half life, as specifically disclosed in U.S. Ser. No. 12/341,769,
hereby incorporated by reference in its entirety, including, but
not limited to, 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F,
436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L.
H. Ablation Variants
[0254] Similarly, another category of functional variants are
"Fc.gamma.R ablation variants" or "Fc knock out (FcKO or KO)"
variants. In these embodiments, for some therapeutic applications,
it is desirable to reduce or remove the normal binding of the Fc
domain to one or more or all of the Fc.gamma. receptors (e.g.
Fc.gamma.R1, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIIa, etc.) to
avoid additional mechanisms of action. That is, for example, in
many embodiments, particularly in the use of bispecific
immunomodulatory antibodies desirable to ablate Fc.gamma.RIIIa
binding to eliminate or significantly reduce ADCC activity such
that one of the Fc domains comprises one or more Fc.gamma. receptor
ablation variants. These ablation variants are depicted in FIG. 6,
and each can be independently and optionally included or excluded,
with preferred aspects utilizing ablation variants selected from
the group consisting of G236R/L328R,
E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,
E233P/L234V/L235A/G236del/S239K/A327G,
E233P/L234V/L235A/G236del/S267K/A327G and
E233P/L234V/L235A/G236del. It should be noted that the ablation
variants referenced herein ablate Fc.gamma.R binding but generally
not FcRn binding.
I. Combination of Heterodimeric and Fc Variants
[0255] As will be appreciated by those in the art, all of the
recited heterodimerization variants (including skew and/or pI
variants) can be optionally and independently combined in any way,
as long as they retain their "strandedness" or "monomer partition".
In addition, all of these variants can be combined into any of the
heterodimerization formats.
[0256] In the case of pI variants, while embodiments finding
particular use are shown in the Figures, other combinations can be
generated, following the basic rule of altering the pI difference
between two monomers to facilitate purification.
[0257] In addition, any of the heterodimerization variants, skew
and pI, are also independently and optionally combined with Fc
ablation variants, Fc variants, FcRn variants, as generally
outlined herein.
VII. Useful Formats of the Invention
[0258] As will be appreciated by those in the art and discussed
more fully below, the bispecific heterodimeric antibodies of the
present invention can take on a wide variety of configurations, as
are generally depicted in FIG. 2. Some figures depict "single
ended" configurations, where there is one type of specificity on
one "arm" of the molecule and a different specificity on the other
"arm". Other figures depict "dual ended" configurations, where
there is at least one type of specificity at the "top" of the
molecule and one or more different specificities at the "bottom" of
the molecule. Thus, the present invention is directed to novel
immunoglobulin compositions that co-engage a different first and a
second antigen.
[0259] As will be appreciated by those in the art, the
heterodimeric formats (see FIG. 2) of the invention can have
different valencies as well as be bispecific. That is, antibodies
of the invention can be bivalent and bispecific, wherein a
checkpoint target is bound by one ABD and the costimulatory target
is bound by a second ABD (see for example the bottle opener format
which is heterodimeric) or the bispecific mAb which is
homodimeric). The heterodimeric antibodies can also be trivalent
and bispecific, wherein the first antigen is bound by two ABDs and
the second antigen by a second ABD (see for example the
Central-scFv format and the trident format). The heterodimeric
antibodies can also be bispecific and tetravalent (such as the
Central scFv2 format and the DVD-Ig format).
[0260] Again, with the exception of the DVD-Ig format and the
central-scFv2 format, the antibodies are generally formatted such
that the co-stimulatory target is bound monovalently.
A. Bottle Opener Format
[0261] One heterodimeric scaffold that finds particular use in the
present invention is the "triple F" or "bottle opener" scaffold
format. In this embodiment, one heavy chain of the antibody
contains a single chain Fv ("scFv", as defined below) and the other
heavy chain is a "regular" Fab format, comprising a variable heavy
chain and a light chain. This structure is sometimes referred to
herein as "triple F" format (scFv-Fab-Fc) or the "bottle-opener"
format, due to a rough visual similarity to a bottle-opener. The
two chains are brought together by the use of amino acid variants
in the constant regions (e.g. the Fc domain, the CH1 domain and/or
the hinge region) that promote the formation of heterodimeric
antibodies as is described more fully below.
[0262] There are several distinct advantages to the present "triple
F" format. As is known in the art, antibody analogs relying on two
scFv constructs often have stability and aggregation problems,
which can be alleviated in the present invention by the addition of
a "regular" heavy and light chain pairing. In addition, as opposed
to formats that rely on two heavy chains and two light chains,
there is no issue with the incorrect pairing of heavy and light
chains (e.g. heavy 1 pairing with light 2, etc.).
[0263] Many of the embodiments outlined herein rely in general on
the bottle opener format that comprises a first monomer comprising
an scFv, comprising a variable heavy and a variable light domain,
covalently attached using an scFv linker (charged, in many but not
all instances), where the scFv is covalently attached to the
N-terminus of a first Fc domain usually through a domain linker
(which, as outlined herein can either be un-charged or charged and
can be exogeneous or endogeneous (e.g. all or part of the native
hinge domain). The second monomer of the bottle opener format is a
heavy chain, and the composition further comprises a light
chain.
[0264] In addition, the Fc domains of the bottle opener format
generally comprise skew variants (e.g. a set of amino acid
substitutions as shown in FIG. 4, with particularly useful skew
variants being selected from the group consisting of
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,
K370S:S364K/E357Q, T366S/L368A/Y407V: T366W and
T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants
(including those shown in FIG. 6), optionally charged scFv linkers
(including those shown in FIG. 8) and the heavy chain comprises pI
variants (including those shown in FIG. 5).
[0265] In some embodiments, the bottle opener format includes skew
variants, pI variants, and ablation variants. Accordingly, some
embodiments include bottle opener formats that comprise: a) a first
monomer (the "scFv monomer") that comprises a charged scFv linker
(with the +H sequence of FIG. 8 being preferred in some
embodiments), the skew variants S364K/E357Q, the ablation variants
E233P/L234V/L235A/G236del/S267K, and an Fv that binds to one target
as outlined herein; b) a second monomer (the "Fab monomer") that
comprises the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a variable heavy domain that,
with the variable light domain, makes up an Fv that binds to a
second target as outlined herein; and c) a light chain. In this
particular embodiment, suitable monomer Fv pairs include (Fabs
listed first, scFvs second) ICOS.times.PD-1, ICOS.times.PD-L1,
ICOS.times.CTLA-4, ICOS.times.LAG-3, ICOS.times.TIM-3,
ICOS.times.BTLA, ICOS.times.TIGIT, TIGIT.times.ICOS,
PD-1.times.ICOS, PD-L1.times.ICOS, CTLA-4.times.ICOS,
LAG-3.times.ICOS, TIM-3.times.ICOS, BTLA.times.ICOS,
OX40.times.TIGIT, OX40.times.PD-1, OX40.times.PD-L1,
OX40.times.CTLA-4, OX40.times.LAG-3, OX40.times.TIM-3,
OX40.times.BTLA, TIGIT.times.OX40, PD-1.times.OX40,
PD-L1.times.OX40, CTLA-4.times.OX40, LAG-3.times.OX40,
TIM-3.times.OX40, BTLA.times.OX40, GITR.times.TIGIT,
GITR.times.PD-1, GITR.times.PD-L1, GITR.times.CTLA-4,
GITR.times.LAG-3, GITR.times.TIM-3, GITR.times.BTLA,
TIGIT.times.GITR, PD-1.times.GITR, PD-L1.times.GITR,
CTLA-4.times.GITR, LAG-3.times.GITR, TIM-3.times.GITR,
BTLA.times.GITR, 4-1BB.times.TIGIT, 4-1BB.times.PD-1,
4-1BB.times.PD-L1, 4-1BB.times.CTLA-4, 4-1BB.times.LAG-3,
4-1BB.times.TIM-3, 4-1BB.times.BTLA, TIGIT.times.4-1BB,
PD-1.times.4-1BB, PD-L1.times.4-1BB, CTLA-4.times.4-1BB,
LAG-3.times.4-1BB, TIM-3.times.4-1BB and BTLA.times.4-1BB.
[0266] Accordingly, some embodiments include bottle opener formats
that comprise: a) a first monomer (the "scFv monomer") that
comprises a charged scFv linker (with the +H sequence of FIG. 8
being preferred in some embodiments), the skew variants
S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K,
and an Fv that binds to one target as outlined herein; b) a second
monomer (the "Fab monomer") that comprises the skew variants
L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the
ablation variants E233P/L234V/L235A/G236del/S267K, and a variable
heavy domain that, with the variable light domain, makes up an Fv
that binds to a second target as outlined herein; and c) a light
chain. In some particular embodiments with these variants in this
format:
[0267] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0268] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0269] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0270] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0271] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0272] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0273] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0274] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0275] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0276] In some embodiments, the bottle opener format includes skew
variants, pI variants, ablation variants and FcRn variants.
Accordingly, some embodiments include bottle opener formats that
comprise: a) a first monomer (the "scFv monomer") that comprises a
charged scFv linker (with the +H sequence of FIG. 8 being preferred
in some embodiments), the skew variants S364K/E357Q, the ablation
variants E233P/L234V/L235A/G236del/S267K, the FcRn variants
M428L/N434S and an Fv that binds to a first receptor (either a
costimulatory or checkpoint receptor) as outlined herein; b) a
second monomer (the "Fab monomer") that comprises the skew variants
L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the
ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn
variants M428L/N434S and a variable heavy domain that, with the
variable light domain, makes up an Fv that binds to a second
receptor as outlined herein (the other of the costimulatory or
checkpoint receptor); and c) a light chain. In some particular
embodiments with these variants in this format:
[0277] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0278] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0279] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0280] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0281] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0282] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0283] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0284] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0285] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0286] Specifically, FIG. 9 shows some bottle opener "backbone"
sequences that are missing the Fv sequences that can be used in the
present invention. That is, Fv sequences for the scFv portion and
the Fab portion can be used from any combination of ICOS and PD-1,
ICOS and CTLA-4, ICOS and LAG-3, ICOS and TIM-3, ICOS and PD-L1,
ICOS and BTLA, ICOS and TIGIT, GITR and TIGIT, GITR and PD-1, GITR
and CTLA-4, GITR and LAG-3, GITR and TIM-3, GITR and PD-L1, GITR
and BTLA, OX40 and PD-1, OX40 and TIGIT, OX40 and CTLA-4, IC OX40
OS and LAG-3, OX40 and TIM-3, OX40 and PD-L1, OX40 and BTLA, 4-1BB
and PD-1, 4-1BB and CTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3, 4-1BB
and PD-L1, TIGIT and 4-1BB and 4-1BB and BTLA, in combination with
any or all of backbones 1 to 10, with backbone 1 of particular use
in these combinations.
[0287] For bottle opener backbone 1 from FIG. 9 (optionally
including the 428L/434S variants) specific Fv combinations of use
in the present invention include ICOS and PD-1, ICOS and PD-L1 and
ICOS and CTLA-4.
[0288] For bottle opener backbone 1 from FIG. 9 (optionally
including the 428L/434S variants) specific ABDs that bind human
ICOS include, but are not limited to, [ICOS]_H0L0 and
[ICOS]H0.66_L0 and and those shown in FIG. 19, FIGS. 20A-20G, FIG.
24, FIGS. 68A-68G and FIGS. 77A-77BA-77B as well as SEQ ID NO:
27869-28086, 28087-28269, 27193-27335, 28549-28556 and
28557-28665.
[0289] For bottle opener backbone 1 from FIG. 9 (optionally
including the 428L/434S variants), specific ABDs that bind human
GITR include, but are not limited to, those in FIG. 18, FIG. 72 and
FIG. 73 and those listed in SEQ ID NO:26282-26290.
[0290] For bottle opener backbone 1 from FIG. 9 (optionally
including the 428L/434S variants), specific ABDs that bind OX40
include, but are not limited to, FIG. 17, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO: 26272-26281.
[0291] For bottle opener backbone 1 from FIG. 9 (optionally
including the 428L/434S variants), specific ABDs that bind human
4-1BB include, but are not limited to, FIG. 16, FIG. 72 and FIG. 73
and SEQ ID NO: 26262-2671.
[0292] For bottle opener backbone 1 from FIG. 9 (optionally
including the 428L/434S variants), specific ABDs that bind human
PD-L1 include, but are not limited to, FIG. 15A-15C, FIG. 73 and
FIG. 78 and SEQ ID NO: 3961-4432.
[0293] Specific bottle opener embodiments are outlined below.
B. mAb-Fv Format
[0294] One heterodimeric scaffold that finds particular use in the
present invention is the mAb-Fv format. In this embodiment, the
format relies on the use of a C-terminal attachment of an "extra"
variable heavy domain to one monomer and the C-terminal attachment
of an "extra" variable light domain to the other monomer, thus
forming a third antigen binding domain (i.e. an "extra" Fv domain),
wherein the Fab portions of the two monomers bind one checkpoint
target and the "extra" Fv domain binds a costimulatory target.
[0295] In this embodiment, the first monomer comprises a first
heavy chain, comprising a first variable heavy domain and a first
constant heavy domain comprising a first Fc domain, with a first
variable light domain covalently attached to the C-terminus of the
first Fc domain using a domain linker
(vhl-CH1-hinge-CH2-CH3-[optional linker]-vl2). The second monomer
comprises a second variable heavy domain, a second constant heavy
domain comprising a second Fc domain, and a third variable heavy
domain covalently attached to the C-terminus of the second Fc
domain using a domain linker (vhl-CH1-hinge-CH2-CH3-[optional
linker]-vh2. This embodiment further utilizes a common light chain
comprising a variable light domain and a constant light domain,
which associates with the heavy chains to form two identical Fabs
that include two identical Fvs. The two C-terminally attached
variable domains make up the "extra" third Fv. As for many of the
embodiments herein, these constructs include skew variants, pI
variants, ablation variants, additional Fc variants, etc. as
desired and described herein. In this embodiment, suitable Fv pairs
include (Fabs listed first, "extra" Fv listed second)
ICOS.times.PD-1, ICOS.times.PD-L1, ICOS.times.CTLA-4,
ICOS.times.LAG-3, ICOS.times.TIM-3, ICOS.times.BTLA,
ICOS.times.TIGIT, TIGIT.times.ICOS, PD-1.times.ICOS,
PD-L1.times.ICOS, CTLA-4.times.ICOS, LAG-3.times.ICOS,
TIM-3.times.ICOS, BTLA.times.ICOS, OX40.times.TIGIT,
OX40.times.PD-1, OX40.times.PD-L1, OX40.times.CTLA-4,
OX40.times.LAG-3, OX40.times.TIM-3, OX40.times.BTLA,
TIGIT.times.OX40, PD-1.times.OX40, PD-L1.times.OX40,
CTLA-4.times.OX40, LAG-3.times.OX40, TIM-3.times.OX40,
BTLA.times.OX40, GITR.times.TIGIT, GITR.times.PD-1,
GITR.times.PD-L1, GITR.times.CTLA-4, GITR.times.LAG-3,
GITR.times.TIM-3, GITR.times.BTLA, TIGIT.times.GITR,
PD-1.times.GITR, PD-L1.times.GITR, CTLA-4.times.GITR,
LAG-3.times.GITR, TIM-3.times.GITR, BTLA.times.GITR,
4-1BB.times.TIGIT, 4-1BB.times.PD-1, 4-1BB.times.PD-L1,
4-1BB.times.CTLA-4, 4-1BB.times.LAG-3, 4-1BB.times.TIM-3,
4-1BB.times.BTLA, TIGIT.times.4-1BB, PD-1.times.4-1BB,
PD-L1.times.4-1BB, CTLA-4.times.4-1BB, LAG-3.times.4-1BB,
TIM-3.times.4-1BB and BTLA.times.4-1BB.
[0296] The ABD sequences for these combinations can be as disclosed
in the sequence listing or as shown in the Figures.
[0297] In addition, the Fc domains of the mAb-Fv format comprise
skew variants (e.g. a set of amino acid substitutions as shown in
FIG. 4, with particularly useful skew variants being selected from
the group consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K;
L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W
and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation
variants (including those shown in FIG. 6), optionally charged scFv
linkers (including those shown in FIG. 8) and the heavy chain
comprises pI variants (including those shown in FIG. 5).
[0298] In some embodiments, the mAb-Fv format includes skew
variants, pI variants, and ablation variants. Accordingly, some
embodiments include mAb-Fv formats that comprise: a) a first
monomer that comprises the skew variants S364K/E357Q, the ablation
variants E233P/L234V/L235A/G236del/S267K, and a first variable
heavy domain that, with the first variable light domain of the
light chain, makes up an Fv that binds to a first checkpoint
inhibitor, and a second variable heavy domain; b) a second monomer
that comprises the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain
that, with the first variable light domain, makes up the Fv that
binds to the first receptor (either a costimulatory receptor or a
checkpoint receptor) as outlined herein, and a second variable
light chain, that together with the second variable heavy chain
forms an Fv (ABD) that binds a second receptor (e.g. the other of
the costimulatory or checkpoint receptor; and c) a light chain
comprising a first variable light domain and a constant light
domain. Of particular use in some embodiments in this format, are
(Fab-scFv order) ICOS.times.PD-1, PD-1.times.ICOS,
PD-L1.times.ICOS, ICOS.times.PD-L1, GITR.times.PD-1,
OX40.times.PD-1 and 4-1BB.times.PD-1. In some particular
embodiments with these variants in this format:
[0299] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0300] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0301] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0302] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0303] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0304] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0305] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0306] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0307] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0308] In some embodiments, the mAb-Fv format includes skew
variants, pI variants, ablation variants and FcRn variants.
Accordingly, some embodiments include mAb-Fv formats that comprise:
a) a first monomer that comprises the skew variants S364K/E357Q,
the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn
variants M428L/N434S and a first variable heavy domain that, with
the first variable light domain of the light chain, makes up an Fv
that binds to a first checkpoint inhibitor, and a second variable
heavy domain; b) a second monomer that comprises the skew variants
L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the
ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn
variants M428L/N434S and a first variable heavy domain that, with
the first variable light domain, makes up the Fv that binds to the
first checkpoint inhibitor as outlined herein, and a second
variable light chain, that together with the second variable heavy
chain forms an Fv (ABD) that binds a second checkpoint inhibitors;
and c) a light chain comprising a first variable light domain and a
constant light domain. Of particular use in some embodiments in
this format, are (Fab-scFv order) ICOS.times.PD-1,
ICOS.times.PD-L1, GITR.times.PD-1, OX40.times.PD-1 and
4-1BB.times.PD-1.
[0309] For mAb-Fv sequences that are similar to the mAb-scFv
backbone 1 (optionally including M428L/N434S) from FIG. 75,
specific ABDs that bind human ICOS are [ICOS]_H0L0 and
[ICOS]_H0.66_L0, as well as those shown in FIG. 19, FIGS. 20A-20G,
FIG. 24, FIGS. 68A-68G and FIGS. 77A-77B as well as SEQ ID NO:
27869-28086, 28087-28269, 27193-27335, 28549-28556 and
28557-28665.
[0310] For mAb-Fv sequences that are similar to the mAb-scFv
backbone 1 (optionally including M428L/N434S) from FIG. 75,
specific ABDs that bind human PD-L1 are shown in FIG. 15A-15C, FIG.
73 and FIG. 78 and SEQ ID NO: 3961-4432.
[0311] For mAb-Fv sequences that are similar to the mAb-scFv
backbone 1 (optionally including M428L/N434S) from FIG. 75,
specific ABDs that bind human GITR are those in FIG. 18, FIG. 72
and FIG. 73 and those listed in SEQ ID NO:26282-26290.
[0312] For mAb-Fv sequences that are similar to the mAb-scFv
backbone 1 (optionally including M428L/N434S) from FIG. 75,
specific ABDs that bind human 4-1BB are those in FIG. 16, FIG. 72
and FIG. 73 and SEQ ID NO: 26262-2671.
[0313] For mAb-Fv sequences that are similar to the mAb-scFv
backbone 1 (optionally including M428L/N434S) from FIG. 75,
specific ABDs that bind human OX40 are those in FIG. 17, FIG. 72
and FIG. 73 and those listed in SEQ ID NO: 26272-26281.
[0314] For mAb-Fv sequences that are similar to the mAb-scFv
backbone 1 (optionally including M428L/N434S) from FIG. 75,
specific ABDs that bind human PD-L1 from FIG. 15A-15C, FIG. 73 and
FIG. 78 and SEQ ID NO: 3961-4432.
C. mAb-scFv
[0315] One heterodimeric scaffold that finds particular use in the
present invention is the mAb-scFv format. In this embodiment, the
format relies on the use of a C-terminal attachment of an scFv to
one of the monomers, thus forming a third antigen binding domain,
wherein the Fab portions of the two monomers bind one receptor
target and the "extra" scFv domain binds the other receptor target
(generally the monovalently bound costimulatory receptor).
[0316] In this embodiment, the first monomer comprises a first
heavy chain (comprising a variable heavy domain and a constant
domain), with a C-terminally covalently attached scFv comprising a
scFv variable light domain, an scFv linker and a scFv variable
heavy domain in either orientation (vhl-CH1-hinge-CH2-CH3-[optional
linker]-vh2-scFv linker-vl2 or vhl-CH1-hinge-CH2-CH3-[optional
linker]-vl2-scFvlinker-vh2). This embodiment further utilizes a
common light chain comprising a variable light domain and a
constant light domain, which associates with the heavy chains to
form two identical Fabs that bind one of the target receptors. As
for many of the embodiments herein, these constructs include skew
variants, pI variants, ablation variants, additional Fc variants,
etc. as desired and described herein. In this embodiment, suitable
Fv pairs include (Fabs listed first, scFvs second) ICOS.times.PD-1,
ICOS.times.PD-L1, ICOS.times.CTLA-4, ICOS.times.LAG-3,
ICOS.times.TIM-3, ICOS.times.BTLA, ICOS.times.TIGIT,
TIGIT.times.ICOS, PD-1.times.ICOS, PD-L1.times.ICOS,
CTLA-4.times.ICOS, LAG-3.times.ICOS, TIM-3.times.ICOS,
BTLA.times.ICOS, OX40.times.TIGIT, OX40.times.PD-1,
OX40.times.PD-L1, OX40.times.CTLA-4, OX40.times.LAG-3,
OX40.times.TIM-3, OX40.times.BTLA, TIGIT.times.OX40,
PD-1.times.OX40, PD-L1.times.OX40, CTLA-4.times.OX40,
LAG-3.times.OX40, TIM-3.times.OX40, BTLA.times.OX40,
GITR.times.TIGIT, GITR.times.PD-1, GITR.times.PD-L1,
GITR.times.CTLA-4, GITR.times.LAG-3, GITR.times.TIM-3,
GITR.times.BTLA, TIGIT.times.GITR, PD-1.times.GITR,
PD-L1.times.GITR, CTLA-4.times.GITR, LAG-3.times.GITR,
TIM-3.times.GITR, BTLA.times.GITR, 4-1BB.times.TIGIT,
4-1BB.times.PD-1, 4-1BB.times.PD-L1, 4-1BB.times.CTLA-4,
4-1BB.times.LAG-3, 4-1BB.times.TIM-3, 4-1BB.times.BTLA,
TIGIT.times.4-1BB, PD-1.times.4-1BB, PD-L1.times.4-1BB,
CTLA-4.times.4-1BB, LAG-3.times.4-1BB, TIM-3.times.4-1BB and
BTLA.times.4-1BB.
[0317] The ABD sequences for these combinations can be as disclosed
in the sequence listing or as shown in the Figures.
[0318] In addition, the Fc domains of the mAb-scFv format generally
comprise skew variants (e.g. a set of amino acid substitutions as
shown in FIG. 4, with particularly useful skew variants being
selected from the group consisting of S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;
L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W
and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation
variants (including those shown in FIG. 6), optionally charged scFv
linkers (including those shown in FIG. 8) and the heavy chain
comprises pI variants (including those shown in FIG. 5).
[0319] In some embodiments, the mAb-scFv format includes skew
variants, pI variants, and ablation variants. Accordingly, some
embodiments include bottle opener formats that comprise: a) a first
monomer that comprises the skew variants S364K/E357Q, the ablation
variants E233P/L234V/L235A/G236del/S267K, and a first variable
heavy domain that, with the first variable light domain of the
light chain, makes up an Fv that binds to a first receptor, and a
scFv that binds to the second receptor; b) a second monomer that
comprises the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain
that, with the first variable light domain, makes up the Fv that
binds to the first receptor as outlined herein; and c) a light
chain comprising a first variable light domain and a constant light
domain. In some particular embodiments with these variants in this
format:
[0320] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0321] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0322] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0323] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0324] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0325] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0326] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0327] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0328] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0329] In some embodiments, the mAb-scFv format includes skew
variants, pI variants, ablation variants and FcRn variants.
Accordingly, some embodiments include mAb-scFv formats that
comprise: a) a first monomer that comprises the skew variants
S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K,
the FcRn variants M428L/N434S and a first variable heavy domain
that, with the first variable light domain of the light chain,
makes up an Fv that binds to a first receptor, and a scFv that
binds to the second receptor; b) a second monomer that comprises
the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and
a first variable heavy domain that, with the first variable light
domain, makes up the Fv that binds to the first checkpoint
inhibitor as outlined herein, and c) a light chain comprising a
first variable light domain and a constant light domain.
[0330] In mAb-scFv backbone 1 (optionally including M428L/N434S)
from FIG. 75, specific ABDs that bind human ICOS are shown in FIG.
19, FIGS. 20A-20G, FIG. 24, FIG. 68
[0331] A-68G and FIGS. 77A-77B as well as SEQ ID NO: 27869-28086,
28087-28269, 27193-27335, 28549-28556 and 28557-28665.
[0332] In mAb-scFv backbone 1 (optionally including M428L/N434S)
from FIG. 75, specific ABDs that bind human GITR are those in FIG.
18, FIG. 72 and FIG. 73 and those listed in SEQ ID
NO:26282-26290.
[0333] In mAb-scFv backbone 1 (optionally including M428L/N434S)
from FIG. 75, specific ABDs that bind human 4-1BB include those in
FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.
[0334] In mAb-scFv backbone 1 (optionally including M428L/N434S)
from FIG. 75, specific ABDs that bind human OX-40 FIG. 17, FIG. 72
and FIG. 73 and those listed in SEQ ID NO: 26272-26281.
[0335] In mAb-scFv backbone 1 (optionally including M428L/N434S)
from FIG. 75, specific ABDs that bind human PD-L1 include FIG.
15A-15C, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.
D. Central scFv
[0336] One heterodimeric scaffold that finds particular use in the
present invention is the Central-scFv format. In this embodiment,
the format relies on the use of an inserted scFv domain thus
forming a third antigen binding domain, wherein the Fab portions of
the two monomers bind one receptor target and the "extra" scFv
domain binds another (again, generally the costimulatory receptor
is bound monovalently). The scFv domain is inserted between the Fc
domain and the CH1-Fv region of one of the monomers, thus providing
a third antigen binding domain.
[0337] In this embodiment, one monomer comprises a first heavy
chain comprising a first variable heavy domain, a CH1 domain (and
optional hinge) and Fc domain, with a scFv comprising a scFv
variable light domain, an scFv linker and a scFv variable heavy
domain. The scFv is covalently attached between the C-terminus of
the CH1 domain of the heavy constant domain and the N-terminus of
the first Fc domain using optional domain linkers
(vhl-CH1-[optional linker]-vh2-scFv linker-vl2-[optional linker
including the hinge]-CH2-CH3, or the opposite orientation for the
scFv, vhl-CH1-[optional linker]-vl2-scFv linker-vh2-[optional
linker including the hinge]-CH2-CH3). In some embodiments, the
optional linker is a hinge or fragment thereof. The other monomer
is a standard Fab side (e.g. vhl-CH1-hinge-CH2-CH3). This
embodiment further utilizes a common light chain comprising a
variable light domain and a constant light domain, which associates
with the heavy chains to form two identical Fabs that bind a
checkpoint inhibitor. As for many of the embodiments herein, these
constructs include skew variants, pI variants, ablation variants,
additional Fc variants, etc. as desired and described herein. In
this embodiment, suitable Fv pairs include (Fabs listed first,
scFvs second) ICOS.times.PD-1, ICOS.times.PD-L1, ICOS.times.CTLA-4,
ICOS.times.LAG-3, ICOS.times.TIM-3, ICOS.times.BTLA,
ICOS.times.TIGIT, TIGIT.times.ICOS, PD-1.times.ICOS,
PD-L1.times.ICOS, CTLA-4.times.ICOS, LAG-3.times.ICOS,
TIM-3.times.ICOS, BTLA.times.ICOS, OX40.times.TIGIT,
OX40.times.PD-1, OX40.times.PD-L1, OX40.times.CTLA-4,
OX40.times.LAG-3, OX40.times.TIM-3, OX40.times.BTLA,
TIGIT.times.OX40, PD-1.times.OX40, PD-L1.times.OX40,
CTLA-4.times.OX40, LAG-3.times.OX40, TIM-3.times.OX40,
BTLA.times.OX40, GITR.times.TIGIT, GITR.times.PD-1,
GITR.times.PD-L1, GITR.times.CTLA-4, GITR.times.LAG-3,
GITR.times.TIM-3, GITR.times.BTLA, TIGIT.times.GITR,
PD-1.times.GITR, PD-L1.times.GITR, CTLA-4.times.GITR,
LAG-3.times.GITR, TIM-3.times.GITR, BTLA.times.GITR,
4-1BB.times.TIGIT, 4-1BB.times.PD-1, 4-1BB.times.PD-L1,
4-1BB.times.CTLA-4, 4-1BB.times.LAG-3, 4-1BB.times.TIM-3,
4-1BB.times.BTLA, TIGIT.times.4-1BB, PD-1.times.4-1BB,
PD-L1.times.4-1BB, CTLA-4.times.4-1BB, LAG-3.times.4-1BB,
TIM-3.times.4-1BB and BTLA.times.4-1BB.
[0338] The ABD sequences for these combinations can be as disclosed
in the sequence listing or as shown in the Figures.
[0339] In addition, the Fc domains of the central scFv format
generally comprise skew variants (e.g. a set of amino acid
substitutions as shown in FIG. 4, with particularly useful skew
variants being selected from the group consisting of
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,
K370S:S364K/E357Q, T366S/L368A/Y407V:T366W and
T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants
(including those shown in FIG. 6), optionally charged scFv linkers
(including those shown in FIG. 8) and the heavy chain comprises pI
variants (including those shown in FIG. 5).
[0340] In some embodiments, the central scFv format includes skew
variants, pI variants, and ablation variants. Accordingly, some
embodiments include central scFv formats that comprise: a) a first
monomer that comprises the skew variants S364K/E357Q, the ablation
variants E233P/L234V/L235A/G236del/S267K, and a first variable
heavy domain that, with the first variable light domain of the
light chain, makes up an Fv that binds to a first receptor; b) a
second monomer that comprises the skew variants L368D/K370S, the pI
variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain
that, with the first variable light domain, makes up the Fv that
binds to the second receptor as outlined herein; and c) a light
chain comprising a first variable light domain and a constant light
domain. In some particular embodiments with these variants in this
format:
[0341] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0342] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0343] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0344] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0345] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0346] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0347] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0348] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0349] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0350] In some embodiments, the central scFv format includes skew
variants, pI variants, ablation variants and FcRn variants.
Accordingly, some embodiments include central-scFv formats that
comprise: a) a first monomer that comprises the skew variants
S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K,
the FcRn variants M428L/N434S and a first variable heavy domain
that, with the first variable light domain of the light chain,
makes up an Fv that binds to a first receptor and an scFv domain
that binds to a second receptor; b) a second monomer that comprises
the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and
a first variable heavy domain that, with the first variable light
domain, makes up the Fv that binds to the first checkpoint
inhibitor as outlined herein; and c) a light chain comprising a
first variable light domain and a constant light domain. In this
embodiment, suitable Fv pairs include (Fabs listed first, scFvs
second) ICOS.times.PD-1, PD-1.times.ICOS, ICOS.times.PD-L1,
PD-L1.times.ICOS, ICOS.times.CTLA-4 and CTLA-4.times.ICOS.
[0351] For central-scFv sequences that utilize the central-scFv
sequences of FIG. 55 suitable Fvs that bind ICOS include, but are
not limited to, shown in FIG. 19, FIGS. 20A-20G, FIG. 24, FIGS.
68A-68G and FIGS. 77A-77B as well as SEQ ID NO: 27869-28086,
28087-28269, 27193-27335, 28549-28556 and 28557-28665.
[0352] For central-scFv sequences that utilize the central-scFv
sequences of FIG. 55 suitable Fvs that bind PD-L1 include, but are
not limited to, those shown in FIG. 15A-15C, FIG. 73 and FIG. 78
and SEQ ID NO: 3961-4432.
[0353] For central-scFv sequences that utilize the central-scFv
sequences of FIG. 55 suitable Fvs that bind GITR include, but are
not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and those
listed in SEQ ID NO:26282-26290.
[0354] For central-scFv sequences that utilize the central-scFv
sequences of FIG. 55 suitable Fvs that bind OX40 include, but are
not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in
SEQ ID NO: 26272-26281.
[0355] For central-scFv sequences that utilize the central-scFv
sequences of FIG. 55 suitable Fvs that bind 4-1BB include, but are
not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
E. Central-scFv2
[0356] One heterodimeric scaffold that finds particular use in the
present invention is the Central-scFv2 format, which is bispecific
and tetravalent. In this embodiment, the format relies on the use
of two inserted scFv domains thus forming third and fourth antigen
binding domains, wherein the Fab portions of the two monomers bind
one receptor target and the "extra" scFv domains bind another. The
scFv domain is inserted between the Fc domain and the CH1-Fv region
of the monomers.
[0357] In this embodiment, both monomers comprise a first heavy
chain comprising a first variable heavy domain, a CH1 domain (and
optional hinge) and Fc domain, with a scFv comprising a scFv
variable light domain, an scFv linker and a scFv variable heavy
domain. The scFv is covalently attached between the C-terminus of
the CH1 domain of the heavy constant domain and the N-terminus of
the first Fc domain using optional domain linkers
(vhl-CH1-[optional linker]-vh2-scFv linker-vl2-[optional linker
including the hinge]-CH2-CH3, or the opposite orientation for the
scFv, vhl-CH1-[optional linker]-vl2-scFv linker-vh2-[optional
linker including the hinge]-CH2-CH3). In some embodiments, the
optional linker is a hinge or fragment thereof. This embodiment
further utilizes a common light chain comprising a variable light
domain and a constant light domain, which associates with the heavy
chains to form two identical Fabs that bind a receptor. As for many
of the embodiments herein, these constructs include skew variants,
pI variants, ablation variants, additional Fc variants, etc. as
desired and described herein. In this embodiment, suitable Fv pairs
include (Fabs listed first, scFvs second) ICOS.times.PD-1,
ICOS.times.PD-L1, ICOS.times.CTLA-4, ICOS.times.LAG-3,
ICOS.times.TIM-3, ICOS.times.BTLA, ICOS.times.TIGIT,
TIGIT.times.ICOS, PD-1.times.ICOS, PD-L1.times.ICOS,
CTLA-4.times.ICOS, LAG-3.times.ICOS, TIM-3.times.ICOS,
BTLA.times.ICOS, OX40.times.TIGIT, OX40.times.PD-1,
OX40.times.PD-L1, OX40.times.CTLA-4, OX40.times.LAG-3,
OX40.times.TIM-3, OX40.times.BTLA, TIGIT.times.OX40,
PD-1.times.OX40, PD-L1.times.OX40, CTLA-4.times.OX40,
LAG-3.times.OX40, TIM-3.times.OX40, BTLA.times.OX40,
GITR.times.TIGIT, GITR.times.PD-1, GITR.times.PD-L1,
GITR.times.CTLA-4, GITR.times.LAG-3, GITR.times.TIM-3,
GITR.times.BTLA, TIGIT.times.GITR, PD-1.times.GITR,
PD-L1.times.GITR, CTLA-4.times.GITR, LAG-3.times.GITR,
TIM-3.times.GITR, BTLA.times.GITR, 4-1BB.times.TIGIT,
4-1BB.times.PD-1, 4-1BB.times.PD-L1, 4-1BB.times.CTLA-4,
4-1BB.times.LAG-3, 4-1BB.times.TIM-3, 4-1BB.times.BTLA,
TIGIT.times.4-1BB, PD-1.times.4-1BB, PD-L1.times.4-1BB,
CTLA-4.times.4-1BB, LAG-3.times.4-1BB, TIM-3.times.4-1BB and
BTLA.times.4-1BB.
[0358] The ABD sequences for these combinations can be as disclosed
in the sequence listing or as shown in the Figures.
[0359] In addition, the Fc domains of the central scFv2 format
generally comprise skew variants (e.g. a set of amino acid
substitutions as shown in FIG. 4, with particularly useful skew
variants being selected from the group consisting of S364K/E357Q:
L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,
K370S:S364K/E357Q, T366S/L368A/Y407V:T366W and
T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants
(including those shown in FIG. 6), optionally charged scFv linkers
(including those shown in FIG. 8) and the heavy chain comprises pI
variants (including those shown in FIG. 5).
[0360] In some embodiments, the central scFv2 format includes skew
variants, pI variants, and ablation variants. Accordingly, some
embodiments include central scFv formats that comprise: a) a first
monomer that comprises the skew variants S364K/E357Q, the ablation
variants E233P/L234V/L235A/G236del/S267K, and a first variable
heavy domain that, with the first variable light domain of the
light chain, makes up an Fv that binds to a first receptor; b) a
second monomer that comprises the skew variants L368D/K370S, the pI
variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain
that, with the first variable light domain, makes up the Fv that
binds to the second receptor as outlined herein; and c) a light
chain comprising a first variable light domain and a constant light
domain. In some particular embodiments with these variants in this
format:
[0361] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0362] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0363] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0364] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0365] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0366] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0367] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0368] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0369] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0370] In some embodiments, the central scFv2 format includes skew
variants, pI variants, ablation variants and FcRn variants.
Accordingly, some embodiments include central-scFv formats that
comprise: a) a first monomer that comprises the skew variants
S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K,
the FcRn variants M428L/N434S and a first variable heavy domain
that, with the first variable light domain of the light chain,
makes up an Fv that binds to a first receptor and an scFv domain
that binds to a second receptor; b) a second monomer that comprises
the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and
a first variable heavy domain that, with the first variable light
domain, makes up the Fv that binds to the first checkpoint
inhibitor as outlined herein; and c) a light chain comprising a
first variable light domain and a constant light domain. In this
embodiment, suitable Fv pairs include (Fabs listed first, scFvs
second) ICOS.times.PD-1, PD-1.times.ICOS, ICOS.times.PD-L1,
PD-L1.times.ICOS, ICOS.times.CTLA-4 and CTLA-4.times.ICOS.
[0371] For central-scFv2 sequences that utilize the central-scFv2
sequences of FIG. 55 suitable Fvs that bind ICOS include, but are
not limited to, shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS.
68A-68G and FIGS. 77A-77B as well as SEQ ID NO: 27869-28086,
28087-28269, 27193-27335, 28549-28556 and 28557-28665.
[0372] For central-scFv2 sequences that utilize the central-scFv2
sequences of FIG. 55 suitable Fvs that bind PD-L1 include, but are
not limited to, those shown in FIG. 15A-15C, FIG. 73 and FIG. 78
and SEQ ID NO: 3961-4432.
[0373] For central-scFv2 sequences that utilize the central-scFv
sequences of FIG. 55 suitable Fvs that bind GITR include, but are
not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and those
listed in SEQ ID NO:26282-26290.
[0374] For central-scFv2 sequences that utilize the central-scFv
sequences of FIG. 55 suitable Fvs that bind OX40 include, but are
not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in
SEQ ID NO: 26272-26281.
[0375] For central-scFv2 sequences that utilize the central-scFv
sequences of FIG. 55 suitable Fvs that bind 4-1BB include, but are
not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
F. One Armed mAb
[0376] As noted above, surprisingly and unexpectedly, monovalent
costimulatory antibodies comprising a single ABD to the target show
efficacy in activating T cells.
[0377] Accordingly, in some embodiments, the invention provides
monovalent, monospecific antibodies as shown in Figure FIG. 2N that
comprise a heterodimeric Fc domain (for stability). In this
embodiment, in this embodiment, one monomer comprises just an Fc
domain, while the other monomer is a HC (VH1-CH1-hinge-CH2-CH3).
This embodiment further utilizes a light chain comprising a
variable light domain and a constant light domain, that associates
with the heavy chain to form a Fab. As for many of the embodiments
herein, these constructs include skew variants, pI variants,
ablation variants, additional Fc variants, etc. as desired and
described herein. In this embodiment, suitable ABDs bind a
costimulatory receptor such as ICOS, GITR, OX40 or 4-1BB.
[0378] The ABD sequences for these combinations can be as disclosed
in the sequence listing or as shown in the Figures.
[0379] In addition, the Fc domains of the comprise skew variants
(e.g. a set of amino acid substitutions as shown in FIG. 4, with
particularly useful skew variants being selected from the group
consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K;
L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W
and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation
variants (including those shown in FIG. 6), and the heavy chain
comprises pI variants (including those shown in FIG. 5).
[0380] In some embodiments, the one armed scFv-mAb format includes
skew variants, pI variants, and ablation variants. Accordingly,
some embodiments include formats that comprise: a) a first (Fc)
monomer that comprises the skew variants S364K/E357Q, the ablation
variants E233P/L234V/L235A/G236del/S267K b) a second monomer that
comprises the skew variants L368D/K370S, the pI variants
Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain
that, with the first variable light domain, makes up the Fv that
binds to the costimulatory receptor as outlined herein
[0381] In this format, specific ABDs that bind human ICOS include,
but are not limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and
those shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS. 68A-68G and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665.
[0382] In this format, specific ABDs that bind human GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0383] In this format, specific ABDs that bind OX40 include, but
are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed
in SEQ ID NO: 26272-26281.
[0384] In this format, specific ABDs that bind human 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0385] In this format, specific ABDs that bind human PD-L1 include,
but are not limited to, FIG. 15A-15C, FIG. 73 and FIG. 78 and SEQ
ID NO: 3961-4432.
[0386] Specific "one armed mAbs" are shown in the Figures and
sequence listing.
G. Bispecific mAb
[0387] One heterodimeric scaffold that finds particular use in the
present invention is the bispecific mAb format, which is bispecific
and tetravalent. In this embodiment, the format relies on the
generation of separate homodimeric antibodies which are then
recombined. In this format, there is one HC-LC pair
(VH1-CH1-hinge-CH2-CH3 and VL1-LC) and a second Hc-LC pair
(VH2-CH1-hinge-CH2-CH3 and VL2-LC), e.g. two different heavy chains
and two different light chains. Reference is made to Example
5I(d).
[0388] In this format, suitable pairs include ICOS and PD-1, ICOS
and CTLA-4, ICOS and LAG-3, ICOS and TIM-3, ICOS and PD-L1, ICOS
and BTLA, ICOS and TIGIT, GITR and TIGIT, GITR and PD-1, GITR and
CTLA-4, GITR and LAG-3, GITR and TIM-3, GITR and PD-L1, GITR and
BTLA, OX40 and PD-1, OX40 and TIGIT, OX40 and CTLA-4, IC OX40 OS
and LAG-3, OX40 and TIM-3, OX40 and PD-L1, OX40 and BTLA, 4-1BB and
PD-1, 4-1BB and CTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3, 4-1BB and
PD-L1, TIGIT and 4-1BB and 4-1BB and BTLA.
[0389] In some particular embodiments with these variants in this
format:
[0390] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0391] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the ABD comprising the ABD 1G6_L1.194_H1.279 that binds to
PD-1;
[0392] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the ABD H3.23_L0.129 that binds to CTLA-4;
[0393] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0394] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0395] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0396] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0397] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0398] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0399] In this format, specific ABDs that bind human ICOS include,
but are not limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and
those shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS. 68A-68G and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665.
[0400] In this format, specific ABDs that bind human GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0401] In this format, specific ABDs that bind OX40 include, but
are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed
in SEQ ID NO: 26272-26281.
[0402] In this format, specific ABDs that bind human 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0403] In this format, specific ABDs that bind human PD-L1 include,
but are not limited to, FIG. 15A-15C, FIG. 73 and FIG. 78 and SEQ
ID NO: 3961-4432.
H. Central-Fv Format
[0404] One heterodimeric scaffold that finds particular use in the
present invention is the Central-Fv format shown in FIG. 2. In this
embodiment, the format relies on the use of an inserted Fv domain
thus forming an "extra" third antigen binding domain, wherein the
Fab portions of the two monomers bind one receptor and the "extra"
central-Fv domain binds another (generally the costimulatory
receptor). The Fv domain is inserted between the Fc domain and the
CH1-Fv region of the monomers, thus providing a third antigen
binding domain, wherein each monomer contains a component of the Fv
(e.g. one monomer comprises a variable heavy domain and the other a
variable light domain of the "extra" central Fv domain).
[0405] In this embodiment, one monomer comprises a first heavy
chain comprising a first variable heavy domain, a CH1 domain, and
Fc domain and an additional variable light domain. The additional
variable light domain is covalently attached between the C-terminus
of the CH1 domain of the heavy constant domain and the N-terminus
of the first Fc domain using domain linkers (vhl-CH1-[optional
linker]-vl2-hinge-CH2-CH3). The other monomer comprises a first
heavy chain comprising a first variable heavy domain, a CH1 domain
and Fc domain and an additional variable heavy domain
(vhl-CH1-[optional linker]-vh2-hinge-CH2-CH3). The additional
variable heavy domain domain is covalently attached between the
C-terminus of the CH1 domain of the heavy constant domain and the
N-terminus of the first Fc domain using domain linkers. This
embodiment utilizes a common light chain comprising a variable
light domain and a constant light domain, that associates with the
heavy chains to form two identical Fabs that each bind a receptor.
The additional variable heavy domain and additional variable light
domain form an "extra" central Fv that binds a second receptor. As
for many of the embodiments herein, these constructs include skew
variants, pI variants, ablation variants, additional Fc variants,
etc. as desired and described herein. In this embodiment, suitable
Fv pairs include (Fabs listed first, "extra" central Fv second)
ICOS.times.PD-1, ICOS.times.PD-L1, ICOS.times.CTLA-4,
ICOS.times.LAG-3, ICOS.times.TIM-3, ICOS.times.BTLA,
ICOS.times.TIGIT, TIGIT.times.ICOS, PD-1.times.ICOS,
PD-L1.times.ICOS, CTLA-4.times.ICOS, LAG-3.times.ICOS,
TIM-3.times.ICOS, BTLA.times.ICOS, OX40.times.TIGIT,
OX40.times.PD-1, OX40.times.PD-L1, OX40.times.CTLA-4,
OX40.times.LAG-3, OX40.times.TIM-3, OX40.times.BTLA,
TIGIT.times.OX40, PD-1.times.OX40, PD-L1.times.OX40,
CTLA-4.times.OX40, LAG-3.times.OX40, TIM-3.times.OX40,
BTLA.times.OX40, GITR.times.TIGIT, GITR.times.PD-1,
GITR.times.PD-L1, GITR.times.CTLA-4, GITR.times.LAG-3,
GITR.times.TIM-3, GITR.times.BTLA, TIGIT.times.GITR,
PD-1.times.GITR, PD-L1.times.GITR, CTLA-4.times.GITR,
LAG-3.times.GITR, TIM-3.times.GITR, BTLA.times.GITR,
4-1BB.times.TIGIT, 4-1BB.times.PD-1, 4-1BB.times.PD-L1,
4-1BB.times.CTLA-4, 4-1BB.times.LAG-3, 4-1BB.times.TIM-3,
4-1BB.times.BTLA, TIGIT.times.4-1BB, PD-1.times.4-1BB,
PD-L1.times.4-1BB, CTLA-4.times.4-1BB, LAG-3.times.4-1BB,
TIM-3.times.4-1BB and BTLA.times.4-1BB.
[0406] The ABD sequences for these combinations can be as disclosed
in the sequence listing or as shown in the Figures.
[0407] In addition, the Fc domains of the central-Fv format
generally comprise skew variants (e.g. a set of amino acid
substitutions as shown in FIG. 4, with particularly useful skew
variants being selected from the group consisting of
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,
K370S:S364K/E357Q, T366S/L368A/Y407V:T366W and
T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants
(including those shown in FIG. 6), optionally charged scFv linkers
(including those shown in FIG. 8) and the heavy chain comprises pI
variants (including those shown in FIG. 5).
[0408] In some embodiments, the central-Fv format includes skew
variants, pI variants, and ablation variants. Accordingly, some
embodiments include central scFv formats that comprise: a) a first
monomer that comprises the skew variants S364K/E357Q, the ablation
variants E233P/L234V/L235A/G236del/S267K, and a first variable
heavy domain that, with the first variable light domain of the
light chain, makes up an Fv that binds to a first receptor; b) a
second monomer that comprises the skew variants L368D/K370S, the pI
variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain
that, with the first variable light domain, makes up the Fv that
binds to the second receptor as outlined herein; and c) a light
chain comprising a first variable light domain and a constant light
domain. In some particular embodiments with these variants in this
format:
[0409] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0410] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0411] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0412] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0413] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0414] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0415] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0416] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0417] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0418] In some embodiments, the central-Fv format includes skew
variants, pI variants, ablation variants and FcRn variants.
Accordingly, some embodiments include central-scFv formats that
comprise: a) a first monomer that comprises the skew variants
S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K,
the FcRn variants M428L/N434S and a first variable heavy domain
that, with the first variable light domain of the light chain,
makes up an Fv that binds to a first receptor and an scFv domain
that binds to a second receptor; b) a second monomer that comprises
the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and
a first variable heavy domain that, with the first variable light
domain, makes up the Fv that binds to the first checkpoint
inhibitor as outlined herein; and c) a light chain comprising a
first variable light domain and a constant light domain. In this
embodiment, suitable Fv pairs include (Fabs listed first, scFvs
second) ICOS.times.PD-1, PD-1.times.ICOS, ICOS.times.PD-L1,
PD-L1.times.ICOS, ICOS.times.CTLA-4 and CTLA-4.times.ICOS.
[0419] For central-Fv formats suitable Fvs that bind ICOS include,
but are not limited to, shown in FIG. 19, FIGS. 20A-20G, FIG. 24,
FIGS. 68A-68G and FIGS. 77A-77B as well as SEQ ID NO: 27869-28086,
28087-28269, 27193-27335, 28549-28556 and 28557-28665.
[0420] For central-Fv formats suitable Fvs that bind PD-L1 include,
but are not limited to, those shown in FIG. 15A-15C, FIG. 73 and
FIG. 78 and SEQ ID NO: 3961-4432.
[0421] For central-Fv formats suitable Fvs that bind GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0422] For central-Fv formats suitable Fvs that bind OX40 include,
but are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those
listed in SEQ ID NO: 26272-26281.
[0423] For central-Fv formats suitable Fvs that bind 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0424] For central-FV formats suitable Fvs that bind PD-L1 include,
but are not limited to, those of FIG. 15A-15C, FIG. 73 and FIG. 78
and SEQ ID NO: 3961-4432.
I. One Armed Central-scFv
[0425] One heterodimeric scaffold that finds particular use in the
present invention is the one armed central-scFv format shown in
FIG. 1C. In this embodiment, one monomer comprises just an Fc
domain, while the other monomer includes a Fab domain (a first
antigen binding domain), a scFv domain (a second antigen binding
domain) and an Fc domain, where the scFv domain is inserted between
the Fc domain and the Fc domain. In this format, the Fab portion
binds one receptor target and the scFv binds another.
[0426] In this embodiment, one monomer comprises a first heavy
chain comprising a first variable heavy domain, a CH1 domain and Fc
domain, with a scFv comprising a scFv variable light domain, an
scFv linker and a scFv variable heavy domain. The scFv is
covalently attached between the C-terminus of the CH1 domain of the
heavy constant domain and the N-terminus of the first Fc domain
using domain linkers, in either orientation, VH1-CH1-[optional
domain linker]-VH2-scFv linker-VL2-[optional domain linker]-CH2-CH3
or VH1-CH1-[optional domain linker]-VL2-scFv linker-VH2-[optional
domain linker]-CH2-CH3. The second monomer comprises an Fc domain
(CH2-CH3). This embodiment further utilizes a light chain
comprising a variable light domain and a constant light domain,
that associates with the heavy chain to form a Fab. As for many of
the embodiments herein, these constructs include skew variants, pI
variants, ablation variants, additional Fc variants, etc. as
desired and described herein. In this embodiment, suitable Fv pairs
include (Fabs listed first, scFvs second) ICOS.times.PD-1,
ICOS.times.PD-L1, ICOS.times.CTLA-4, ICOS.times.LAG-3,
ICOS.times.TIM-3, ICOS.times.BTLA, ICOS.times.TIGIT,
TIGIT.times.ICOS, PD-1.times.ICOS, PD-L1.times.ICOS,
CTLA-4.times.ICOS, LAG-3.times.ICOS, TIM-3.times.ICOS,
BTLA.times.ICOS, OX40.times.TIGIT, OX40.times.PD-1,
OX40.times.PD-L1, OX40.times.CTLA-4, OX40.times.LAG-3,
OX40.times.TIM-3, OX40.times.BTLA, TIGIT.times.OX40,
PD-1.times.OX40, PD-L1.times.OX40, CTLA-4.times.OX40,
LAG-3.times.OX40, TIM-3.times.OX40, BTLA.times.OX40,
GITR.times.TIGIT, GITR.times.PD-1, GITR.times.PD-L1,
GITR.times.CTLA-4, GITR.times.LAG-3, GITR.times.TIM-3,
GITR.times.BTLA, TIGIT.times.GITR, PD-1.times.GITR,
PD-L1.times.GITR, CTLA-4.times.GITR, LAG-3.times.GITR,
TIM-3.times.GITR, BTLA.times.GITR, 4-1BB.times.TIGIT,
4-1BB.times.PD-1, 4-1BB.times.PD-L1, 4-1BB.times.CTLA-4,
4-1BB.times.LAG-3, 4-1BB.times.TIM-3, 4-1BB.times.BTLA,
TIGIT.times.4-1BB, PD-1.times.4-1BB, PD-L1.times.4-1BB,
CTLA-4.times.4-1BB, LAG-3.times.4-1BB, TIM-3.times.4-1BB and
BTLA.times.4-1BB.
[0427] The ABD sequences for these combinations can be as disclosed
in the sequence listing or as shown in the Figure.
[0428] In addition, the Fc domains of the one armed central-scFv
format generally comprise skew variants (e.g. a set of amino acid
substitutions as shown in FIG. 4, with particularly useful skew
variants being selected from the group consisting of
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L,
K370S:S364K/E357Q, T366S/L368A/Y407V:T366W and
T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants
(including those shown in FIG. 6), optionally charged scFv linkers
(including those shown in FIG. 8) and the heavy chain comprises pI
variants (including those shown in FIG. 5).
[0429] In some embodiments, the one armed central-scFv format
includes skew variants, pI variants, and ablation variants.
Accordingly, some embodiments include formats that comprise: a) a
first monomer that comprises the skew variants S364K/E357Q, the
ablation variants E233P/L234V/L235A/G236del/S267K, and a variable
heavy domain that, with the variable light domain of the light
chain, makes up an Fv that binds to a first receptor, and a scFv
that binds to the other receptor; b) a second monomer that
comprises the skew variants L368D/K370S, the pI variants
Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and c) a light chain comprising a
first variable light domain and a constant light domain. In some
particular embodiments with these variants in this format:
[0430] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0431] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0432] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0433] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0434] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0435] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0436] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0437] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0438] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0439] In some embodiments, the one armed central-scFv format
includes skew variants, pI variants, ablation variants and FcRn
variants. Accordingly, some embodiments include formats that
comprise: a) a first monomer that comprises the skew variants
S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K,
the FcRn variants M428L/N434S and a first variable heavy domain
that, with the first variable light domain of the light chain,
makes up an Fv that binds to a first receptor and a scFv domain
that binds to a second receptor; b) a second monomer that comprises
the skew variants L368D/K370S, the pI variants
Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S. and
c) a light chain comprising a first variable light domain and a
constant light domain.
[0440] In this format, specific ABDs that bind human ICOS include,
but are not limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and
those shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS. 68A-68G and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665.
[0441] In this format, specific ABDs that bind human GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0442] In this format, specific ABDs that bind OX40 include, but
are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed
in SEQ ID NO: 26272-26281.
[0443] In this format, specific ABDs that bind human 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0444] In this format, specific ABDs that bind human PD-L1 include,
but are not limited to, FIG. 15A-15C, FIG. 73 and FIG. 78 and SEQ
ID NO: 3961-4432.
J. One Armed scFv-mAb
[0445] One heterodimeric scaffold that finds particular use in the
present invention is the one armed scFv-mAb format shown in FIG.
2D. In this embodiment, one monomer comprises just an Fc domain,
while the other monomer uses a scFv domain attached at the
N-terminus of the heavy chain, generally through the use of a
linker: vhl-scFv linker-vl1-[optional domain
linker]-VH2-CH1-hinge-CH2-CH3 or (in the opposite orientation)
vl1-scFv linker-vh1-[optional domain linker]-VH2-CH1-hinge-CH2-CH3.
In this format, either the Fab portion binds one receptor target
and the scFv binds another. This embodiment further utilizes a
light chain comprising a variable light domain and a constant light
domain, that associates with the heavy chain to form a Fab. As for
many of the embodiments herein, these constructs include skew
variants, pI variants, ablation variants, additional Fc variants,
etc. as desired and described herein. In this embodiment, suitable
Fv pairs include (Fabs listed first, scFvs second) ICOS.times.PD-1,
ICOS.times.PD-L1, ICOS.times.CTLA-4, ICOS.times.LAG-3,
ICOS.times.TIM-3, ICOS.times.BTLA, ICOS.times.TIGIT,
TIGIT.times.ICOS, PD-1.times.ICOS, PD-L1.times.ICOS,
CTLA-4.times.ICOS, LAG-3.times.ICOS, TIM-3.times.ICOS,
BTLA.times.ICOS, OX40.times.TIGIT, OX40.times.PD-1,
OX40.times.PD-L1, OX40.times.CTLA-4, OX40.times.LAG-3,
OX40.times.TIM-3, OX40.times.BTLA, TIGIT.times.OX40,
PD-1.times.OX40, PD-L1.times.OX40, CTLA-4.times.OX40,
LAG-3.times.OX40, TIM-3.times.OX40, BTLA.times.OX40,
GITR.times.TIGIT, GITR.times.PD-1, GITR.times.PD-L1,
GITR.times.CTLA-4, GITR.times.LAG-3, GITR.times.TIM-3,
GITR.times.BTLA, TIGIT.times.GITR, PD-1.times.GITR,
PD-L1.times.GITR, CTLA-4.times.GITR, LAG-3.times.GITR,
TIM-3.times.GITR, BTLA.times.GITR, 4-1BB.times.TIGIT,
4-1BB.times.PD-1, 4-1BB.times.PD-L1, 4-1BB.times.CTLA-4,
4-1BB.times.LAG-3, 4-1BB.times.TIM-3, 4-1BB.times.BTLA,
TIGIT.times.4-1BB, PD-1.times.4-1BB, PD-L1.times.4-1BB,
CTLA-4.times.4-1BB, LAG-3.times.4-1BB, TIM-3.times.4-1BB and
BTLA.times.4-1BB.
[0446] The ABD sequences for these combinations can be as disclosed
in the sequence listing or as shown in the Figures.
[0447] In addition, the Fc domains of the comprise skew variants
(e.g. a set of amino acid substitutions as shown in FIG. 4, with
particularly useful skew variants being selected from the group
consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K;
L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W
and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation
variants (including those shown in FIG. 6), optionally charged scFv
linkers (including those shown in FIG. 8) and the heavy chain
comprises pI variants (including those shown in FIG. 5).
[0448] In some embodiments, the one armed scFv-mAb format includes
skew variants, pI variants, and ablation variants. Accordingly,
some embodiments include formats that comprise: a) a first monomer
that comprises the skew variants S364K/E357Q, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain
that, with the first variable light domain of the light chain,
makes up an Fv that binds to a first checkpoint inhibitor, and a
second variable heavy domain; b) a second monomer that comprises
the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain
that, with the first variable light domain, makes up the Fv that
binds to the first checkpoint inhibitor as outlined herein, and a
second variable light chain, that together with the second variable
heavy chain forms an Fv (ABD) that binds a second checkpoint
inhibitors; and c) a light chain comprising a first variable light
domain and a constant light domain. In some particular embodiments
with these variants in this format:
[0449] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0450] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0451] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0452] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303_L1.34 that binds to LAG-3;
[0453] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0454] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0455] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0456] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0457] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0458] In some embodiments, the one armed scFv-mAb format includes
skew variants, pI variants, ablation variants and FcRn variants.
Accordingly, some embodiments include bottle opener formats that
comprise: a) a first monomer that comprises the skew variants
S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K,
the FcRn variants M428L/N434S and a first variable heavy domain
that, with the first variable light domain of the light chain,
makes up an Fv that binds to a first checkpoint inhibitor, and a
second variable heavy domain; b) a second monomer that comprises
the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and
a first variable heavy domain that, with the first variable light
domain, makes up the Fv that binds to the first checkpoint
inhibitor as outlined herein, and a second variable light chain,
that together with the second variable heavy chain forms an Fv
(ABD) that binds a second checkpoint inhibitors; and c) a light
chain comprising a first variable light domain and a constant light
domain.
[0459] In this format, specific ABDs that bind human ICOS include,
but are not limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and
those shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS. 68A-68G and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665.
[0460] In this format, specific ABDs that bind human GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0461] In this format, specific ABDs that bind OX40 include, but
are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed
in SEQ ID NO: 26272-26281.
[0462] In this format, specific ABDs that bind human 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0463] In this format, specific ABDs that bind human PD-L1 include,
but are not limited to, FIG. 15A-15C, FIG. 73 and FIG. 78 and SEQ
ID NO: 3961-4432.
K. scFv-mAb Format
[0464] One heterodimeric scaffold that finds particular use in the
present invention is the mAb-scFv format shown in FIG. 2E. In this
embodiment, the format relies on the use of a N-terminal attachment
of a scFv to one of the monomers, thus forming a third antigen
binding domain, wherein the Fab portions of the two monomers each
bind one target and the "extra" scFv domain binds a different
target.
[0465] In this embodiment, the first monomer comprises a first
heavy chain (comprising a variable heavy domain and a constant
domain), with a N-terminally covalently attached scFv comprising a
scFv variable light domain, an scFv linker and a scFv variable
heavy domain in either orientation ((vhl-scFv linker-vl1-[optional
domain linker]-vh2-CH1-hinge-CH2-CH3) or (with the scFv in the
opposite orientation) ((vl1-scFv linker-vh1-[optional domain
linker]-vh2-CH1-hinge-CH2-CH3)). The second monomer comprises a
heavy chain VH20CH1-hinge-CH2-CH3. This embodiment further utilizes
a common light chain comprising a variable light domain and a
constant light domain, that associates with the heavy chains to
form two identical Fabs that bind one of the target antigens. As
for many of the embodiments herein, these constructs include skew
variants, pI variants, ablation variants, additional Fc variants,
etc. as desired and described herein. In this embodiment, suitable
Fv pairs include (Fabs listed first, scFvs second) ICOS.times.PD-1,
ICOS.times.PD-L1, ICOS.times.CTLA-4, ICOS.times.LAG-3,
ICOS.times.TIM-3, ICOS.times.BTLA, ICOS.times.TIGIT,
TIGIT.times.ICOS, PD-1.times.ICOS, PD-L1.times.ICOS,
CTLA-4.times.ICOS, LAG-3.times.ICOS, TIM-3.times.ICOS,
BTLA.times.ICOS, OX40.times.TIGIT, OX40.times.PD-1,
OX40.times.PD-L1, OX40.times.CTLA-4, OX40.times.LAG-3,
OX40.times.TIM-3, OX40.times.BTLA, TIGIT.times.OX40,
PD-1.times.OX40, PD-L1.times.OX40, CTLA-4.times.OX40,
LAG-3.times.OX40, TIM-3.times.OX40, BTLA.times.OX40,
GITR.times.TIGIT, GITR.times.PD-1, GITR.times.PD-L1,
GITR.times.CTLA-4, GITR.times.LAG-3, GITR.times.TIM-3,
GITR.times.BTLA, TIGIT.times.GITR, PD-1.times.GITR,
PD-L1.times.GITR, CTLA-4.times.GITR, LAG-3.times.GITR,
TIM-3.times.GITR, BTLA.times.GITR, 4-1BB.times.TIGIT,
4-1BB.times.PD-1, 4-1BB.times.PD-L1, 4-1BB.times.CTLA-4,
4-1BB.times.LAG-3, 4-1BB.times.TIM-3, 4-1BB.times.BTLA,
TIGIT.times.4-1BB, PD-1.times.4-1BB, PD-L1.times.4-1BB,
CTLA-4.times.4-1BB, LAG-3.times.4-1BB, TIM-3.times.4-1BB and
BTLA.times.4-1BB.
[0466] The ABD sequences for these combinations can be as disclosed
in the sequence listing or in the Figures.
[0467] In addition, the Fc domains of the scFv-mAb format comprise
skew variants (e.g. a set of amino acid substitutions as shown in
FIG. 4, with particularly useful skew variants being selected from
the group consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K;
L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W
and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation
variants (including those shown in FIG. 6), optionally charged scFv
linkers (including those shown in FIG. 8) and the heavy chain
comprises pI variants (including those shown in FIG. 5).
[0468] In some embodiments, the mAb-scFv format includes skew
variants, pI variants, and ablation variants. Accordingly, some
embodiments include bottle opener formats that comprise: a) a first
monomer that comprises the skew variants S364K/E357Q, the ablation
variants E233P/L234V/L235A/G236del/S267K, and a first variable
heavy domain that, with the first variable light domain of the
light chain, makes up an Fv that binds to a first checkpoint
inhibitor, and a second variable heavy domain; b) a second monomer
that comprises the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain
that, with the first variable light domain, makes up the Fv that
binds to the first checkpoint inhibitor as outlined herein, and a
second variable light chain, that together with the second variable
heavy chain forms an Fv (ABD) that binds a second checkpoint
inhibitors; and c) a light chain comprising a first variable light
domain and a constant light domain. In some particular embodiments
with these variants in this format:
[0469] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0470] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0471] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0472] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0473] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0474] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0475] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0476] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0477] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0478] In some embodiments, the mAb-scFv format includes skew
variants, pI variants, ablation variants and FcRn variants.
Accordingly, some embodiments include bottle opener formats that
comprise: a) a first monomer that comprises the skew variants
S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K,
the FcRn variants M428L/N434S and a first variable heavy domain
that, with the first variable light domain of the light chain,
makes up an Fv that binds to a first checkpoint inhibitor, and a
second variable heavy domain; b) a second monomer that comprises
the skew variants L368D/K370S, the pI variants
N208D/Q295E/N384D/Q418E/N421D, the ablation variants
E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and
a first variable heavy domain that, with the first variable light
domain, makes up the Fv that binds to the first checkpoint
inhibitor as outlined herein, and a second variable light chain,
that together with the second variable heavy chain forms an Fv
(ABD) that binds a second checkpoint inhibitors; and c) a light
chain comprising a first variable light domain and a constant light
domain.
[0479] In this format, specific ABDs that bind human ICOS include,
but are not limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and
those shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS. 68A-68G and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665.
[0480] In this format, specific ABDs that bind human GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0481] In this format, specific ABDs that bind OX40 include, but
are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed
in SEQ ID NO: 26272-26281.
[0482] In this format, specific ABDs that bind human 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0483] In this format, specific ABDs that bind human PD-L1 include,
but are not limited to, FIG. 15A-15C, FIG. 73 and FIG. 78 and SEQ
ID NO: 3961-4432.
L. Dual scFv Formats
[0484] The present invention also provides dual scFv formats as are
known in the art and shown in FIG. 2B. In this embodiment, the
heterodimeric bispecific antibody is made up of two scFv-Fc
monomers (both in either (vh-scFv linker-vl-[optional domain
linker]-CH2-CH3) format or (vl-scFv linker-vh-[optional domain
linker]-CH2-CH3) format, or with one monomer in one orientation and
the other in the other orientation.
[0485] In this case, all ABDs are in the scFv format, with any
combination of ICOS and PD-1, ICOS and CTLA-4, ICOS and LAG-3, ICOS
and TIM-3, ICOS and PD-L1, ICOS and BTLA, GITR and PD-1, GITR and
CTLA-4, GITR and LAG-3, GITR and TIM-3, GITR and PD-L1, GITR and
BTLA, OX40 and PD-1, OX40 and CTLA-4, OX40 and LAG-3, OX40 and
TIM-3, OX40 and PD-L1, OX40 and BTLA, 4-1BB and PD-1, 4-1BB and
CTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3, 4-1BB and PD-L1 and 4-1BB
and BTLA being useful. The ABD sequences for these combinations can
be as disclosed in the sequence listing or as shown in the
Figures.
[0486] In addition, the Fc domains of the dual scFv format comprise
skew variants (e.g. a set of amino acid substitutions as shown in
FIG. 4, with particularly useful skew variants being selected from
the group consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K;
L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W
and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation
variants (including those shown in FIG. 6), optionally charged scFv
linkers (including those shown in FIG. 8) and the heavy chain
comprises pI variants (including those shown in FIG. 5).
[0487] In some embodiments, the dual scFv format includes skew
variants, pI variants, and ablation variants. Accordingly, some
embodiments include formats that comprise: a) a first monomer that
comprises the skew variants S364K/E357Q, the ablation variants
E233P/L234V/L235A/G236del/S267K, and a scFv that binds a first
receptor (VH1-scFv linker-VL1-[optional domain linker]-CH2-CH3 or
VL1-scFv linker-VH1-[optional domain linker]-CH2-CH3) and b) a
first monomer that comprises the skew variants L368D/K370S, the
ablation variants E233P/L234V/L235A/G236del/S267K, and a scFv that
binds a first receptor (VH1-scFv linker-VL1-[optional domain
linker]-CH2-CH3 or VL1-scFv linker-VH1-[optional domain
linker]-CH2-CH3). pI variants can be as outlined herein, but most
common will be charged scFv linkers of opposite charge for each
monomer. FcRn variants, particularly 428L/434S, can optionally be
included. In some particular embodiments with these variants in
this format:
[0488] (1) the format comprises a ABD binds to ICOS that has the
ABD of [ICOS] H0.66_L0;
[0489] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the ABD comprising the ABD 1G6_L1.194_H1.279 that binds to
PD-1;
[0490] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the ABD H3.23_L0.129 that binds to CTLA-4;
[0491] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0492] (5) the format comprises the ABD 1G6_L1.194_H1.279 that
binds to PD-1 and an Fv binds to GITR;
[0493] (6) the format comprises the ABD 1G6_L1.194_H1.279 that
binds to PD-1 and the Fv binds to OX40;
[0494] (7) the format comprises the ABD 1G6_L1.194_H1.279 that
binds to PD-1 and the ABD binds to ICOS;
[0495] 8) the format comprises the ABD 1G6_L1.194_H1.279 that binds
to PD-1 and an ABD binds to 4-1BB; and
[0496] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0497] In this format, specific ABDs that bind human ICOS include,
but are not limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and
those shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS. 68A-68G and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665.
[0498] In this format, specific ABDs that bind human GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0499] In this format, specific ABDs that bind OX40 include, but
are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed
in SEQ ID NO: 26272-26281.
[0500] In this format, specific ABDs that bind human 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0501] In this format, specific ABDs that bind human PD-L1 include,
but are not limited to, FIG. 15A-15C, FIG. 73 and FIG. 78 and SEQ
ID NO: 3961-4432.
M. Non-Heterodimeric Bispecific Antibodies
[0502] As will be appreciated by those in the art, the Fv sequences
outlined herein can also be used in both monospecific antibodies
(e.g. "traditional monoclonal antibodies") or non-heterodimeric
bispecific formats (see FIGS. 2J, K and L).
[0503] In this format, specific ABDs that bind human ICOS include,
but are not limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and
those shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS. 68A-68G and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665.
[0504] In this format, specific ABDs that bind human GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0505] In this format, specific ABDs that bind OX40 include, but
are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed
in SEQ ID NO: 26272-26281.
[0506] In this format, specific ABDs that bind human 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0507] In this format, specific ABDs that bind human PD-L1 include,
but are not limited to, FIG. 15A-15C, FIG. 73 and FIG. 78 and SEQ
ID NO: 3961-4432.
[0508] Suitable non-heterodimeric bispecific formats are known in
the art, and include a number of different formats as generally
depicted in Spiess et al., Molecular Immunology (67):95-106 (2015)
and Kontermann, mAbs 4:2, 182-197 (2012), both of which are
expressly incorporated by reference and in particular for the
figures, legends and citations to the formats therein.
N. DVD-Ig Format
[0509] In some embodiments, the bispecific antibody is in a "Dual
Variable Domain-Ig" or "DVD-Ig.TM." format (see FIG. 2L) such as is
generally described in U.S. Pat. No. 7,612,181, hereby expressly
incorporated by reference in its entirety, and in particular for
the Figures and Legends therein. In the DVD-Ig format, the antibody
is tetravalent and bispecific, and comprises 4 chains: two
homodimeric heavy chains and two identical light chains. The heavy
chains each have a VH1-(optional linker)-VH2-CH1-hinge-CH2-CH3
structure and the two light chains each have a VL1-optional
linker-VL2-CL structure, with VH1 and VL1 forming a first ABD and
the VH2 and VL2 forming a second ABD, where the first and second
ABDs bind a costimulatory and a checkpoint receptor. In this
embodiment, suitable combinations include ICOS and PD-1, ICOS and
CTLA-4, ICOS and LAG-3, ICOS and TIM-3, ICOS and PD-L1, ICOS and
BTLA, GITR and PD-1, GITR and CTLA-4, GITR and LAG-3, GITR and
TIM-3, GITR and PD-L1, GITR and BTLA, OX40 and PD-1, OX40 and
CTLA-4, OX40 and LAG-3, OX40 and TIM-3, OX40 and PD-L1, OX40 and
BTLA, 4-1BB and PD-1, 4-1BB and CTLA-4, 4-1BB and LAG-3, 4-1BB and
TIM-3, 4-1BB and PD-L1 and 4-1BB and BTLA.
[0510] The DVD-Ig.TM. and Central-scFv2 are two formats that are
bispecific and tetravalent, and thus do not bind a costimulatory
receptor in a monovalent fashion. Exemplary DVD-Ig.TM. constructs
are shown in FIG. 61A-61B.
[0511] In some particular embodiments with these variants in this
format:
[0512] (1) the format comprises the ABD of [ICOS]_H0.66_L0;
[0513] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the ABD comprising the ABD 1G6_L1.194_H1.279 that binds to
PD-1;
[0514] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the ABD H3.23_L0.129 that binds to CTLA-4;
[0515] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0516] (5) the format comprises the ABD 1G6_L1.194_H1.279 that
binds to PD-1 and an ABD that binds to GITR;
[0517] (6) the format comprises the ABD 1G6_L1.194_H1.279 that
binds to PD-1 and and ABD that binds to OX40;
[0518] (7) the format comprises the ABD 1G6_L1.194_H1.279 that
binds to PD-1 and an ABD that binds to ICOS;
[0519] 8) the format comprises the ABD 1G6_L1.194_H1.279 that binds
to PD-1 and an ABD that binds to 4-1BB; and
[0520] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0521] In this format, specific ABDs that bind human ICOS include,
but are not limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and
those shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS. 68A-68G and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665.
[0522] In this format, specific ABDs that bind human GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0523] In this format, specific ABDs that bind OX40 include, but
are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed
in SEQ ID NO: 26272-26281.
[0524] In this format, specific ABDs that bind human 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0525] In this format, specific ABDs that bind human PD-L1 include,
but are not limited to, FIG. 15A-15C, FIG. 73 and FIG. 78 and SEQ
ID NO: 3961-4432.
O. Trident Format
[0526] In some embodiments, the bispecific antibodies of the
invention are in the "Trident" format as generally described in
WO2015/184203, hereby expressly incorporated by reference in its
entirety and in particular for the Figures, Legends, definitions
and sequences of "Heterodimer-Promoting Domains" or "HPDs",
including "K-coil" and "E-coil" sequences. Tridents rely on using
two different HPDs that associate to form a heterodimeric structure
as a component of the structure, see FIG. 2M. In this embodiment,
the Trident format include a "traditional" heavy and light chain
(e.g. VH1-CH1-hinge-CH2-CH3 and VL1-CL), a third chain comprising a
first "diabody-type binding domain" or "DART.RTM.",
VH2-(linker)-VL3-HPD1 and a fourth chain comprising a second
DART.RTM., VH3-(linker)-(linker)-VL2-HPD2. The VH1 and VL1 form a
first ABD, the VH2 and VL2 form a second ABD, and the VH3 and VL3
form a third ABD. IN some cases, as is shown in FIG. 2M, the second
and third ABDs bind the same antigen, in this instance generally
the checkpoint receptor, e.g. bivalently, with the first ABD
binding a costimulatory receptor monovalently. In this embodiment,
suitable combinations include ICOS and PD-1, ICOS and CTLA-4, ICOS
and LAG-3, ICOS and TIM-3, ICOS and PD-L1, ICOS and BTLA, GITR and
PD-1, GITR and CTLA-4, GITR and LAG-3, GITR and TIM-3, GITR and
PD-L1, GITR and BTLA, OX40 and PD-1, OX40 and CTLA-4, OX40 and
LAG-3, OX40 and TIM-3, OX40 and PD-L1, OX40 and BTLA, 4-1BB and
PD-1, 4-1BB and CTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3, 4-1BB and
PD-L1 and 4-1BB and BTLA.
[0527] In some particular embodiments with these variants in this
format:
[0528] (1) the format comprises a Fab ABD binds to ICOS that has
the ABD of [ICOS]_H0.66_L0;
[0529] (2) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv ABD comprising the ABD 1G6_L1.194_H1.279 that binds
to PD-1;
[0530] (3) the format comprises an ICOS ABD of H0.66_L0 combined
with the scFv comprising the ABD H3.23_L0.129 that binds to
CTLA-4;
[0531] (4) the format comprises an ICOS ABD of H0.66_L0 is combined
with the ABD 7G8_H.303 L1.34 that binds to LAG-3;
[0532] (5) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to GITR;
[0533] (6) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to OX40;
[0534] (7) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to ICOS;
[0535] 8) the format comprises a scFv comprising the ABD
1G6_L1.194_H1.279 that binds to PD-1 and the Fab binds to 4-1BB;
and
[0536] (9) the format comprises the ABD of H0.66_L0 that binds to
ICOS and an ABD that binds to PD-L1.
[0537] In this format, specific ABDs that bind human ICOS include,
but are not limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and
those shown in FIG. 19, FIG. 20A-20G, FIG. 24, FIGS. 68A-68G and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665.
[0538] In this format, specific ABDs that bind human GITR include,
but are not limited to, those in FIG. 18, FIG. 72 and FIG. 73 and
those listed in SEQ ID NO:26282-26290.
[0539] In this format, specific ABDs that bind OX40 include, but
are not limited to, FIG. 17, FIG. 72 and FIG. 73 and those listed
in SEQ ID NO: 26272-26281.
[0540] In this format, specific ABDs that bind human 4-1BB include,
but are not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:
26262-2671.
[0541] In this format, specific ABDs that bind human PD-L1 include,
but are not limited to, FIG. 15A-15C, FIG. 73 and FIG. 78 and SEQ
ID NO: 3961-4432.
P. Monospecific, Monoclonal Antibodies
[0542] As will be appreciated by those in the art, the novel Fv
sequences outlined herein can also be used in both monospecific
antibodies (e.g. "traditional monoclonal antibodies") or
non-heterodimeric bispecific formats. Accordingly, the present
invention provides monoclonal (monospecific) antibodies comprising
the 6 CDRs and/or the vh and vl sequences from the figures,
generally with IgG1, IgG2, IgG3 or IgG4 constant regions, with
IgG1, IgG2 and IgG4 (including IgG4 constant regions comprising a
S228P amino acid substitution) finding particular use in some
embodiments. That is, any sequence herein with a "H_L" designation
can be linked to the constant region of a human IgG1 antibody.
VIII. Antigen Binding Domains (ABDs) to Target Antigens
[0543] The bispecific antibodies of the invention have two
different antigen binding domains (ABDs) that bind to two different
target receptor antigens ("target pairs"), in either bivalent,
bispecific formats or trivalent, bispecific formats as generally
shown in FIG. 2.
[0544] The bispecific antibodies bind to a first target antigen
comprising a checkpoint receptor and a second target antigen
comprising a costimulatory receptor. Suitable checkpoint receptors
as outlined herein include PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, BTLA
and TIGIT. Suitable costimulatory receptors as outlined herein
include ICOS, GITR, OX40 and 4-1BB. As outlined
[0545] Suitable target checkpoint antigens include human (and
sometimes cyno) PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and BTLA
(sequences in the sequence listing). Accordingly, suitable
bispecific antibodies bind ICOS and PD-1, ICOS and CTLA-4, ICOS and
LAG-3, ICOS and TIM-3, ICOS and PD-L1, ICOS and BTLA, ICOS and
TIGIT, GITR and TIGIT, GITR and PD-1, GITR and CTLA-4, GITR and
LAG-3, GITR and TIM-3, GITR and PD-L1, GITR and BTLA, OX40 and
PD-1, OX40 and TIGIT, OX40 and CTLA-4, IC OX40 OS and LAG-3, OX40
and TIM-3, OX40 and PD-L1, OX40 and BTLA, 4-1BB and PD-1, 4-1BB and
CTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3, 4-1BB and PD-L1, TIGIT
and 4-1BB and 4-1BB and BTLA.
[0546] Note that generally these bispecific antibodies are named
"anti-PD-1.times.anti-CTLA-4", or generally simplistically or for
ease (and thus interchangeably) as "PD-1.times.CTLA-4", etc. for
each pair. Note that unless specified herein, the order of the
antigen list in the name does not confer structure; that is a
PD-1.times.CTLA-4 bottle opener antibody can have the scFv bind to
PD-1 or CTLA-4, although in some cases, the order specifies
structure as indicated.
[0547] As is more fully outlined herein, these combinations of ABDs
can be in a variety of formats, as outlined below, generally in
combinations where one ABD is in a Fab format and the other is in
an scFv format. As discussed herein and shown in FIG. 2, some
formats use a single Fab and a single scFv (A, C and D), and some
formats use two Fabs and a single scFv (E, F, G, H and I).
A. Antigen Binding Domains
[0548] As discussed herein, the bispecific checkpoint heterodimeric
antibodies of the invention include two antigen binding domains
(ABDs), each of which bind to a different checkpoint protein. As
outlined herein, these heterodimeric antibodies can be bispecific
and bivalent (each antigen is bound by a single ABD, for example,
in the format depicted in FIG. 2 A), bispecific and trivalent (one
antigen is bound by a single ABD and the other is bound by two
ABDs, for example as depicted in FIG. 2F, G, H, I or M), or
bispecific and tetravalent (both antigens are bound by two ABDs,
for example as depicted in FIGS. 2J and L).
[0549] In addition, in general, one of the ABDs comprises a scFv as
outlined herein, in an orientation from N- to C-terminus of vh-scFv
linker-vl or vl-scFv linker-vh. One or both of the other ABDs,
according to the format, generally is a Fab, comprising a vh domain
on one protein chain (generally as a component of a heavy chain)
and a vl on another protein chain (generally as a component of a
light chain). Note that the "trident" format uses DART.RTM.s, which
are similar to scFvs except the orientation is different and in
general the linkers can be slightly longer.
[0550] The invention provides a number of ABDs that bind to a
number of different receptor proteins, as outlined below. As will
be appreciated by those in the art, any set of 6 CDRs or vh and vl
domains can be in the scFv format or in the Fab format, which is
then added to the heavy and light constant domains, where the heavy
constant domains comprise variants (including within the CH1 domain
as well as the Fc domain). The scFv sequences contained in the
sequence listing utilize a particular charged linker, but as
outlined herein, uncharged or other charged linkers can be used,
including those depicted in FIG. 8.
[0551] In addition, as discussed above, the numbering used in the
Sequence Listing for the identification of the CDRs is Kabat,
however, different numbering can be used, which will change the
amino acid sequences of the CDRs as shown in Table 1.
[0552] For all of the variable heavy and light domains listed
herein, further variants can be made. As outlined herein, in some
embodiments the set of 6 CDRs can have from 0, 1, 2, 3, 4 or 5
amino acid modifications (with amino acid substitutions finding
particular use), as well as changes in the framework regions of the
variable heavy and light domains, as long as the frameworks
(excluding the CDRs) retain at least about 80, 85 or 90% identity
to a human germline sequence selected from those listed in FIG. 1
of U.S. Pat. No. 7,657,380, which Figure and Legend is incorporated
by reference in its entirety herein. Thus, for example, the
identical CDRs as described herein can be combined with different
framework sequences from human germline sequences, as long as the
framework regions retain at least 80, 85 or 90% identity to a human
germline sequence selected from those listed in FIG. 1 of U.S. Pat.
No. 7,657,380. Alternatively, the CDRs can have amino acid
modifications (e.g. from 1, 2, 3, 4 or 5 amino acid modifications
in the set of CDRs (that is, the CDRs can be modified as long as
the total number of changes in the set of 6 CDRs is less than 6
amino acid modifications, with any combination of CDRs being
changed; e.g. there may be one change in vlCDR1, two in vhCDR2,
none in vhCDR3, etc.)), as well as having framework region changes,
as long as the framework regions retain at least 80, 85 or 90%
identity to a human germline sequence selected from those listed in
FIG. 1 of U.S. Pat. No. 7,657,380.
B. PD-1 Antigen Binding Domains
[0553] In some embodiments, one of the ABDs binds PD-1. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences,
are depicted in FIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG.
74, FIG. 76 and SEQ ID NO:1-2392, 3125-3144, 4697-7594 and
4697-21810, and include those sequences in the sequence listing
with the identifiers 1G6_H1.279_L1.194; 1G6_H1.280_L1.224;
1G6_L1.194_H1.279; 1G6_L1.210_H1.288; and 2E9_H1L1.
[0554] As will be appreciated by those in the art, suitable
anti-PD-1 ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the vh and vl sequences of these sequences.
Suitable ABDs can also include the entire vh and vl sequences as
depicted in these sequences and Figures, used as scFvs or as Fabs.
In many of the embodiments herein that contain an Fv to PD-1, it is
the scFv monomer that binds PD-1. As discussed herein, the other of
the target pair when PD-1 is one of the antigens is selected from
ICOS (suitable sequences are shown in FIG. 19, FIGS. 20A-20G, FIG.
24, FIG. 68A-68G and FIGS. 77A-77B as well as SEQ ID NO:
27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665
(which can be scFv sequences, CDR sequence sets or vh and vl
sequences)), GITR, OX40 and 4-1BB.
[0555] Particularly useful ABDs that bind human PD-1 include, but
are not limited to, 1G6_H1.279_L1.194, 1G6_H1.280_L1.224;
1G6_L1.194_H1.279, 1G6_L1.210_H1.288 and 2E9_H1L1.
[0556] Additionally useful vh and vl sequences that bind human PD-1
are shown in FIG. 76.
[0557] In addition to the parental CDR sets disclosed in the
sequence listing that form an ABD to PD-1, the invention provides
variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2,
3, 4 or 5 amino acid changes from the parental CDRs, as long as the
ABD is still able to bind to the target antigen, as measured by at
least one of a BIACORE.RTM., surface plasmon resonance (SPR) and/or
BLI (biolayer interferometry, e.g. OCTET.RTM. assay) assay, with
the latter finding particular use in many embodiments.
[0558] In addition to the parental variable heavy and variable
light domains disclosed herein that form an ABD to PD-1, the
invention provides variant vh and vl domains. In one embodiment,
the variant vh and vl domains each can have from 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid changes from the parental vh and vl
domain, as long as the ABD is still able to bind to the target
antigen, as measured at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments. In another embodiment, the variant vh and vl are
at least 90, 95, 97, 98 or 99% identical to the respective parental
vh or vl, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments.
[0559] Specific preferred embodiments include the 1G6_L1.194_H1.279
anti-PD-1 Fv, in a scFv format, included within any of the bottle
opener format backbones of FIG. 9.
[0560] Specific preferred embodiments include the 1G6_L1.194_H1.279
anti-PD-1 Fv, in a scFv format, included within any of the format
backbones of FIG. 55.
[0561] Specific preferred embodiments include the 1G6_L1.194_H1.279
anti-PD-1 Fv, in a scFv format, included within any of the mAb-scFv
format backbones of FIG. 75.
[0562] Other embodiments utilize any of the anti-PD-1 vh and vl
domain pairs (either as scFvs or Fabs) as shown in FIG. 76 in any
format shown in FIG. 2.
C. CTLA-4 Antigen Binding Domains
[0563] In some embodiments, one of the ABDs binds CTLA-4. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences,
are depicted in SEQ ID NOs: 2393-2414 and 3737-3816, as well as
sequences of particular interest in some embodiments are shown in
FIG. 12 and FIG. 79 and also include those sequences in the
sequence listing with the identifiers [CTLA-4]_H0.25_L0;
[CTLA-4]_H0.26_L0; [CTLA-4]_H0.27_L0; [CTLA-4]_H0.29_L0;
[CTLA-4]_H0.38_L0; [CTLA-4]_H0.39_L0; 0[CTLA-4]_H0.40_L0;
[CTLA-4]_H0.70_L0; [CTLA-4]_H0_L0.22; [CTLA-4]_H2_L0;
[CTLA-4]_H3.21_L0.124; [CTLA-4]_H3.21_L0.129;
[CTLA-4]_H3.21_L0.132; [CTLA-4]_H3.23_L0.124;
[CTLA-4]_H3.23_L0.129; [CTLA-4]_H3.23_L0.132;
[CTLA-4]_H3.25_L0.124; [CTLA-4]_H3.25_L0.129;
[CTLA-4]_H3.25_L0.132; [CTLA-4]_H3.4_L0.118; [CTLA-4]_H3.4_L0.119;
[CTLA-4]_H3.4_L0.12; [CTLA-4]_H3.4_L0.121; [CTLA-4]_H3.4_L0.122;
[CTLA-4]_H3.4_L0.123; [CTLA-4]_H3.4_L0.124; [CTLA-4]_H3.4_L0.125;
[CTLA-4]_H3.4_L0.126; [CTLA-4]_H3.4_L0.127; [CTLA-4]_H3.4_L0.128;
[CTLA-4]_H3.4_L0.129; [CTLA-4]_H3.4_L0.130; [CTLA-4]_H3.4_L0.131;
[CTLA-4]_H3.4_L0.132; [CTLA-4]_H3.5_L2.1; [CTLA-4]_H3.5_L2.2;
[CTLA-4]_H3.5_L2.3; [CTLA-4]_H3_L0; [CTLA-4]_H3_L0.22;
[CTLA-4]_H3_L0.44; [CTLA-4]_H3_L0.67; and [CTLA-4]_H3_L0.74.
[0564] As will be appreciated by those in the art, suitable
anti-CTLA-4 ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the vh and vl sequences outlined herein.
Suitable ABDs can also include the entire vh and vl sequences as
depicted in these sequences and Figures, used as scFvs or as Fabs.
In many of the embodiments herein that contain an Fv to CTLA-4, it
is the scFv monomer that binds CTLA-4.
[0565] As discussed herein, the other of the target pair when
CTLA-4 is one of the antigens is selected from ICOS (suitable
sequences are shown in FIG. 19, FIGS. 20A-20G, FIG. 24, FIG. 68 and
FIGS. 77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665, and those with the
identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0), GITR, OX40 and
4-1BB.
[0566] In addition to the parental CDR sets disclosed in the
sequence listing that form an ABD to CTLA-4, the invention provides
variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2,
3, 4 or 5 amino acid changes from the parental CDRs, as long as the
ABD is still able to bind to the target antigen, as measured by at
least one of a BIACORE.RTM., surface plasmon resonance (SPR) and/or
BLI (biolayer interferometry, e.g. OCTET.RTM. assay) assay, with
the latter finding particular use in many embodiments.
[0567] In addition to the parental variable heavy and variable
light domains disclosed herein that form an ABD to CTLA-4, the
invention provides variant vh and vl domains. In one embodiment,
the variant vh and vl domains each can have from 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid changes from the parental vh and vl
domain, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments. In another embodiment, the variant vh and vl are
at least 90, 95, 97, 98 or 99% identical to the respective parental
vh or vl, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments.
D. TIM-3 Antigen Binding Domains
[0568] In some embodiments, one of the ABDs binds TIM-3. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences,
are depicted in FIG. 14 and FIG. 81A-81C and SEQ ID NO: 3345-3704,
4585-4696. ABD sequences of particular interest in some embodiments
include those sequences in the sequence listing with the
identifiers 1D10_H0L0; 1D12_H0L0; 3H3_H1_L2.1; 6C8_H0L0;
6D9_H0_1D12_L0; 7A9_H0L0; 7B11_H0L0; 7B11var_H0L0; and
7C2_H0L0.
[0569] As will be appreciated by those in the art, suitable
anti-TIM-3 ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the vh and vl sequences of those depicted
herein. Suitable ABDs can also include the entire vh and vl
sequences as depicted in these sequences and Figures, used as scFvs
or as Fabs. In many of the embodiments herein that contain an Fv to
TIM-3, it is the Fab monomer that binds TIM-3. As discussed herein,
the other of the target pair when TIM-3 is one of the antigens is
selected ICOS (suitable sequences are shown in FIG. 19, FIGS.
20A-20G, FIG. 24, FIGS. 68A-68G and FIGS. 77A-77B as well as SEQ ID
NO: 27869-28086, 28087-28269, 27193-27335, 28549-28556 and
28557-28665, and those with the identifiers [ICOS]_H0.66_L0 and
[ICOS]_H0L0), GITR, OX40 and 4-1BB.
[0570] In addition to the parental CDR sets disclosed in the
sequence listing that form an ABD to TIM-3, the invention provides
variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2,
3, 4 or 5 amino acid changes from the parental CDRs, as long as the
ABD is still able to bind to the target antigen, as measured by at
least one of a BIACORE.RTM., surface plasmon resonance (SPR) and/or
BLI (biolayer interferometry, e.g. OCTET.RTM. assay) assay, with
the latter finding particular use in many embodiments.
[0571] In addition to the parental variable heavy and variable
light domains disclosed herein that form an ABD to TIM-3, the
invention provides variant vh and vl domains. In one embodiment,
the variant vh and vl domains each can have from 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid changes from the parental vh and vl
domain, as long as the ABD is still able to bind to the target
antigen, as measured at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments. In another embodiment, the variant vh and vl are
at least 90, 95, 97, 98 or 99% identical to the respective parental
vh or vl, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments.
[0572] LAG-3 Antigen Binding Domains
[0573] In some embodiments, one of the ABDs binds LAG-3. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences,
are depicted in FIG. 13, FIG. 80 and in SEQ ID NO: 2415-2604,
3817-3960 also include those sequences in the sequence listing with
the identifiers 2A11_H0L0; 2A11_H1.125_L2.113; 2A11_H1.144_L2.142;
2A11_H1_L2.122; 2A11_H1_L2.123; 2A11_H1_L2.124; 2A11_H1_L2.25;
2A11_H1_L2.47; 2A11_H1_L2.50; 2A11_H1_L2.91; 2A11_H1_L2.93;
2A11_H1_L2.97; 2A11_H1L1; 2A11_H1L2; 2A11_H2L2; 2A11_H3L1;
2A11_H3L2; 2A11_H4L1; 2A11_H4L2; 7G8_H0L0; 7G8_H1L1;
7G8_H3.18_L1.11; 7G8_H3.23_L1.11; 7G8_H3.28_L1; 7G8_H3.28_L1.11;
7G8_H3.28_L1.13; 7G8_H3.30_L1.34; 7G8_H3.30_L1.34; and
7G8_H3L1.
[0574] As will be appreciated by those in the art, suitable
anti-LAG-3 ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the vh and vl sequences of the sequences
herein. Suitable ABDs can also include the entire vh and vl
sequences as depicted in these sequences and Figures, used as scFvs
or as Fabs. In many of the embodiments herein that contain an Fv to
LAG-3, it is the Fab monomer that binds LAG-3. As discussed herein,
the other of the target pair when LAG-3 is is one of the antigens
is selected from ICOS (suitable sequences are shown in FIG. 19,
FIGS. 20A-20G, FIG. 24, FIGS. 68A-68G and FIGS. 77A-77B as well as
SEQ ID NO: 27869-28086, 28087-28269, 27193-27335, 28549-28556 and
28557-28665, and those with the identifiers [ICOS]_H0.66_L0 and
[ICOS]_H0L0), GITR, OX40 and 4-1BB.
[0575] In addition to the parental CDR sets disclosed in the
sequence listing that form an ABD to LAG-3, the invention provides
variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2,
3, 4 or 5 amino acid changes from the parental CDRs, as long as the
ABD is still able to bind to the target antigen, as measured by at
least one of a BIACORE.RTM., surface plasmon resonance (SPR) and/or
BLI (biolayer interferometry, e.g. OCTET.RTM. assay) assay, with
the latter finding particular use in many embodiments.
[0576] In addition to the parental variable heavy and variable
light domains disclosed herein that form an ABD to LAG-3, the
invention provides variant vh and vl domains. In one embodiment,
the variant vh and vl domains each can have from 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid changes from the parental vh and vl
domain, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments. In another embodiment, the variant vh and vl are
at least 90, 95, 97, 98 or 99% identical to the respective parental
vh or vl, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments.
E. BTLA Antigen Binding Domains
[0577] In some embodiments, one of the ABDs binds BTLA. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences,
are depicted in FIG. 82, FIG. 84A-84C and SEQ ID NO:3705-3736, and
also include those sequences in the sequence listing with the
identifiers 9C6_H0L0; 9C6_H1.1_L1; and 9C6_H1.11_L1.
[0578] As will be appreciated by those in the art, suitable
anti-BTLA ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the vh and vl sequences depicted herein.
Suitable ABDs can also include the entire vh and vl sequences as
depicted in these sequences and Figures, used as scFvs or as Fabs.
In many of the embodiments herein that contain an Fv to BTLA, it is
the Fab monomer that binds BTLA. As discussed herein, the other of
the target pair when BTLA is is one of the antigens is selected
from ICOS (suitable sequences are shown in FIG. 19, FIGS. 20A-20G,
FIG. 24, FIGS. 68A-68G and FIGS. 77A-77B as well as SEQ ID NO:
27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665,
and those with the identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0),
GITR, OX40 and 4-1BB.
[0579] In addition to the parental CDR sets disclosed in the
sequence listing that form an ABD to BTLA, the invention provides
variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2,
3, 4 or 5 amino acid changes from the parental CDRs, as long as the
ABD is still able to bind to the target antigen, as measured at
least one of a BIACORE.RTM., surface plasmon resonance (SPR) and/or
BLI (biolayer interferometry, e.g. OCTET.RTM. assay) assay, with
the latter finding particular use in many embodiments.
[0580] In addition to the parental variable heavy and variable
light domains disclosed herein that form an ABD to BTLA, the
invention provides variant vh and vl domains. In one embodiment,
the variant vh and vl domains each can have from 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid changes from the parental vh and vl
domain, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments. In another embodiment, the variant vh and vl are
at least 90, 95, 97, 98 or 99% identical to the respective parental
vh or vl, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments.
F. TIGIT Antigen Binding Domains
[0581] In some embodiments, one of the ABDs binds TIGIT. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences,
are depicted in FIG. 8? And in SEQ ID NO:4433-4585.
[0582] As will be appreciated by those in the art, suitable
anti-TIGIT ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the vh and vl sequences depicted herein.
Suitable ABDs can also include the entire vh and vl sequences as
depicted in these sequences and Figures, used as scFvs or as Fabs.
In many of the embodiments herein that contain an Fv to TIGIT, it
is the Fab monomer that binds TIGIT. As discussed herein, the other
of the target pair when TIGIT is is one of the antigens is selected
ICOS (suitable sequences are shown in FIG. 19, FIGS. 20A-20G, FIG.
24, FIGS. 68A-68G and FIGS. 77A-77B as well as SEQ ID NO:
27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665,
and those with the identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0),
GITR, OX40 and 4-1BB.
G. PD-L1 Antigen Binding Domains
[0583] In some embodiments, one of the ABDs binds PD-L1. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences,
are depicted in FIG. 15A-15C, FIG. 73 and FIG. 78, and in SEQ ID
NO:3961-4432.
[0584] As will be appreciated by those in the art, suitable
anti-TIGIT ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the vh and vl sequences depicted herein.
Suitable ABDs can also include the entire vh and vl sequences as
depicted in these sequences and Figures, used as scFvs or as Fabs.
In many of the embodiments herein that contain an Fv to TIGIT, it
is the Fab monomer that binds TIGIT. As discussed herein, the other
of the target pair when TIGIT is is one of the antigens is selected
ICOS (suitable sequences are shown in FIG. 19, FIGS. 20A-20G, FIG.
24, FIGS. 68A-68G and FIGS. 77A-77B as well as SEQ ID NO:
27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665,
and those with the identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0),
GITR, OX40 and 4-1BB.
H. ICOS Antigen Binding Domains
[0585] In some embodiments, one of the ABDs binds ICOS. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences
ICOS (suitable sequences are shown in FIG. 19, FIGS. 20A-20G, FIG.
24, FIGS. 68A-68G and FIGS. 77A-77B as well as SEQ ID NO:
27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665,
and those with the identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0.
[0586] As will be appreciated by those in the art, suitable
anti-ICOS ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the sequences disclosed herein. Suitable
ABDs can also include the entire vh and vl sequences as depicted in
these sequences and Figures, used as scFvs or as Fabs. In many of
the embodiments herein that contain an Fv to ICOS, it is the Fab
monomer that binds ICOS. As discussed herein, the other of the
target pair when ICOS is one of the antigens is selected from PD-1
are depicted in FIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG.
74, FIG. 76 and SEQ ID NO:1-2392, 3125-3144, 4697-7594 and
4697-21810, and include those sequences in the sequence listing
with the identifiers 1G6_H1.279_L1.194; 1G6_H1.280_L1.224;
1G6_L1.194_H1.279; 1G6_L1.210_H1.288; and 2E9_H1L1 (which can be
scFv sequences, CDR sequence sets or vh and vl sequences)), CTLA-4
(suitable sequences are depicted in FIGS. 12, 72 and 79, as well as
SEQ ID NO:2393-2414 and 3737-3816 (which can be scFv sequences, CDR
sequence sets or vh and vl sequences)), TIM-3 (suitable sequences
are depicted in FIG. 14, FIG. 81A-81C and SEQ ID NO: 3345-3704,
4585-4696 (which can be scFv sequences, CDR sequence sets or vh and
vl sequences)), LAG-3 (suitable sequences are depicted in FIG. 13,
FIG. 80 and SEQ ID NO:2415-2604, 3817-3960 (which can be scFv
sequences, CDR sequence sets or vh and vl sequences)), BTLA
(suitable sequences are depicted in FIGS. 82 and 84 and SEQ I
DNO?3705-3736 (which can be scFv sequences, CDR sequence sets or vh
and vl sequences)), and TIGIT (suitable sequences are depicted in
Figure XX and SEQ ID NO:4433-4585 (which can be scFv sequences, CDR
sequence sets or vh and vl sequences)).
[0587] In addition to the parental CDR sets disclosed in the
sequence listing that form an ABD to ICOS, the invention provides
variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2,
3, 4 or 5 amino acid changes from the parental CDRs, as long as the
ABD is still able to bind to the target antigen, as measured at
least one of a BIACORE.RTM., surface plasmon resonance (SPR) and/or
BLI (biolayer interferometry, e.g. OCTET.RTM. assay) assay, with
the latter finding particular use in many embodiments.
[0588] In addition to the parental variable heavy and variable
light domains disclosed herein that form an ABD to ICOS, the
invention provides variant vh and vl domains. In one embodiment,
the variant vh and vl domains each can have from 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid changes from the parental vh and vl
domain, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments. In another embodiment, the variant vh and vl are
at least 90, 95, 97, 98 or 99% identical to the respective parental
vh or vl, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments.
[0589] Specific preferred embodiments include the [ICOS]_H0.66_L0
anti-ICOS Fv, in a Fab format, included within any of the bottle
opener format backbones of FIG. 9.
[0590] Specific preferred embodiments include the [ICOS]_H0_L0
anti-ICOS Fv, in a scFv format, included within any of the bottle
opener format backbones of FIG. 9.
[0591] Specific preferred embodiments include the [ICOS]_H0.66_L0
anti-ICOS Fv, in a scFv format, included within any of the mAb-scFv
format backbones of FIG. 75.
[0592] Specific preferred embodiments include the [ICOS]_H0.66_L0
anti-ICOS Fv, in a Fab format, included within any of the mAb-scFv
format backbones of FIG. 75.
[0593] Specific preferred embodiments include the [ICOS]_H0.66_L0
anti-ICOS Fv, in a Fab format, included within any of the format
backbones of FIG. 55.
I. GITR Antigen Binding Domains
[0594] In some embodiments, one of the ABDs binds GITR. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences
FITR (suitable sequences are shown in FIG. 18, FIG. 72 and FIG. 73,
and in SEQ ID NO: 26282-26290.
[0595] As will be appreciated by those in the art, suitable
anti-GITR ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the sequences disclosed herein. Suitable
ABDs can also include the entire vh and vl sequences as depicted in
these sequences and Figures, used as scFvs or as Fabs. In many of
the embodiments herein that contain an Fv to GITR, it is the Fab
monomer that binds GITR. As discussed herein, the other of the
target pair when GITR is one of the antigens is selected from PD-1
are depicted in FIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG.
74, FIG. 76 and SEQ ID NO:1-2392, 3125-3144, 4697-7594 and
4697-21810, and include those sequences in the sequence listing
with the identifiers 1G6_H1.279_L1.194; 1G6_H1.280_L1.224;
1G6_L1.194_H1.279; 1G6_L1.210_H1.288; and 2E9_H1L1 (which can be
scFv sequences, CDR sequence sets or vh and vl sequences)), CTLA-4
(suitable sequences are depicted in FIGS. 12, 72 and 79, as well as
SEQ ID NO:2393-2414 and 3737-3816 (which can be scFv sequences, CDR
sequence sets or vh and vl sequences)), TIM-3 (suitable sequences
are depicted in FIG. 14, FIG. 81A-81C and SEQ ID NO: 3345-3704,
4585-4696 (which can be scFv sequences, CDR sequence sets or vh and
vl sequences)), LAG-3 (suitable sequences are depicted in FIG. 13,
FIG. 80 and SEQ ID NO:2415-2604, 3817-3960 (which can be scFv
sequences, CDR sequence sets or vh and vl sequences)), BTLA
(suitable sequences are depicted in FIGS. 82 and 84 and SEQ I
DNO?3705-3736 (which can be scFv sequences, CDR sequence sets or vh
and vl sequences)), and TIGIT (suitable sequences are depicted in
Figure XX and SEQ ID NO:4433-4585 (which can be scFv sequences, CDR
sequence sets or vh and vl sequences)).
[0596] In addition to the parental CDR sets disclosed in the
sequence listing that form an ABD to GITR, the invention provides
variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2,
3, 4 or 5 amino acid changes from the parental CDRs, as long as the
ABD is still able to bind to the target antigen, as measured at
least one of a BIACORE.RTM., surface plasmon resonance (SPR) and/or
BLI (biolayer interferometry, e.g. OCTET.RTM. assay) assay, with
the latter finding particular use in many embodiments.
[0597] In addition to the parental variable heavy and variable
light domains disclosed herein that form an ABD to GITR, the
invention provides variant vh and vl domains. In one embodiment,
the variant vh and vl domains each can have from 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid changes from the parental vh and vl
domain, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments. In another embodiment, the variant vh and vl are
at least 90, 95, 97, 98 or 99% identical to the respective parental
vh or vl, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments.
J. OX40 Antigen Binding Domains
[0598] In some embodiments, one of the ABDs binds OX40. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences
for OX40 are provided (suitable sequences are shown in FIG. 17,
FIG. 72 and FIG. 73, and in SEQ ID NO: 26272-26281.
[0599] As will be appreciated by those in the art, suitable
anti-OX40 ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the sequences disclosed herein. Suitable
ABDs can also include the entire vh and vl sequences as depicted in
these sequences and Figures, used as scFvs or as Fabs. In many of
the embodiments herein that contain an Fv to OX40, it is the Fab
monomer that binds GITR. As discussed herein, the other of the
target pair when OX40 is one of the antigens is selected from PD-1
are depicted in FIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG.
74, FIG. 76 and SEQ ID NO:1-2392, 3125-3144, 4697-7594 and
4697-21810, and include those sequences in the sequence listing
with the identifiers 1G6_H1.279_L1.194; 1G6_H1.280_L1.224;
1G6_L1.194_H1.279; 1G6_L1.210_H1.288; and 2E9_H1L1 (which can be
scFv sequences, CDR sequence sets or vh and vl sequences)), CTLA-4
(suitable sequences are depicted in FIGS. 12, 72 and 79, as well as
SEQ ID NO:2393-2414 and 3737-3816 (which can be scFv sequences, CDR
sequence sets or vh and vl sequences)), TIM-3 (suitable sequences
are depicted in FIG. 14, FIG. 81A-81C and SEQ ID NO: 3345-3704,
4585-4696 (which can be scFv sequences, CDR sequence sets or vh and
vl sequences)), LAG-3 (suitable sequences are depicted in FIG. 13,
FIG. 80 and SEQ ID NO:2415-2604, 3817-3960 (which can be scFv
sequences, CDR sequence sets or vh and vl sequences)), BTLA
(suitable sequences are depicted in FIGS. 82 and 84 and SEQ I
DNO?3705-3736 (which can be scFv sequences, CDR sequence sets or vh
and vl sequences)), and TIGIT (suitable sequences are depicted in
Figure XX and SEQ ID NO:4433-4585 (which can be scFv sequences, CDR
sequence sets or vh and vl sequences)).
[0600] In addition to the parental CDR sets disclosed in the
sequence listing that form an ABD to OX40, the invention provides
variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2,
3, 4 or 5 amino acid changes from the parental CDRs, as long as the
ABD is still able to bind to the target antigen, as measured at
least one of a BIACORE.RTM., surface plasmon resonance (SPR) and/or
BLI (biolayer interferometry, e.g. OCTET.RTM. assay) assay, with
the latter finding particular use in many embodiments.
[0601] In addition to the parental variable heavy and variable
light domains disclosed herein that form an ABD to OX40, the
invention provides variant vh and vl domains. In one embodiment,
the variant vh and vl domains each can have from 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid changes from the parental vh and vl
domain, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments. In another embodiment, the variant vh and vl are
at least 90, 95, 97, 98 or 99% identical to the respective parental
vh or vl, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments.
K. 4-1BB Antigen Binding Domains
[0602] In some embodiments, one of the ABDs binds 4-1BB. Suitable
sets of 6 CDRs and/or vh and vl domains, as well as scFv sequences
4-1BB (suitable sequences are shown in FIG. 16, FIG. 72 and FIG.
73, and in SEQ ID NO:26262-2671.
[0603] As will be appreciated by those in the art, suitable
anti-4-1BB ABDs can comprise a set of 6 CDRs as depicted in these
sequences and Figures, either as they are underlined or, in the
case where a different numbering scheme is used as described herein
and as shown in Table 1, as the CDRs that are identified using
other alignments within the sequences disclosed herein. Suitable
ABDs can also include the entire vh and vl sequences as depicted in
these sequences and Figures, used as scFvs or as Fabs. In many of
the embodiments herein that contain an Fv to 4-1BB, it is the Fab
monomer that binds 4-1BB. As discussed herein, the other of the
target pair when ICOS is one of the antigens is selected from PD-1
are depicted in FIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG.
74, FIG. 76 and SEQ ID NO:1-2392, 3125-3144, 4697-7594 and
4697-21810, and include those sequences in the sequence listing
with the identifiers 1G6_H1.279_L1.194; 1G6_H1.280_L1.224;
1G6_L1.194_H1.279; 1G6_L1.210_H1.288; and 2E9_H1L1 (which can be
scFv sequences, CDR sequence sets or vh and vl sequences)), CTLA-4
(suitable sequences are depicted in FIGS. 12, 72 and 79, as well as
SEQ ID NO:2393-2414 and 3737-3816 (which can be scFv sequences, CDR
sequence sets or vh and vl sequences)), TIM-3 (suitable sequences
are depicted in FIG. 14, FIG. 81A-81C and SEQ ID NO: 3345-3704,
4585-4696 (which can be scFv sequences, CDR sequence sets or vh and
vl sequences)), LAG-3 (suitable sequences are depicted in FIG. 13,
FIG. 80 and SEQ ID NO:2415-2604, 3817-3960 (which can be scFv
sequences, CDR sequence sets or vh and vl sequences)), BTLA
(suitable sequences are depicted in FIGS. 82 and 84 and SEQ I
DNO?3705-3736 (which can be scFv sequences, CDR sequence sets or vh
and vl sequences)), and TIGIT (suitable sequences are depicted in
Figure XX and SEQ ID NO:4433-4585 (which can be scFv sequences, CDR
sequence sets or vh and vl sequences)).
[0604] In addition to the parental CDR sets disclosed in the
sequence listing that form an ABD to 4-1BB, the invention provides
variant CDR sets. In one embodiment, a set of 6 CDRs can have 1, 2,
3, 4 or 5 amino acid changes from the parental CDRs, as long as the
ABD is still able to bind to the target antigen, as measured at
least one of a BIACORE.RTM., surface plasmon resonance (SPR) and/or
BLI (biolayer interferometry, e.g. OCTET.RTM. assay) assay, with
the latter finding particular use in many embodiments.
[0605] In addition to the parental variable heavy and variable
light domains disclosed herein that form an ABD to 4-1BB, the
invention provides variant vh and vl domains. In one embodiment,
the variant vh and vl domains each can have from 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid changes from the parental vh and vl
domain, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments. In another embodiment, the variant vh and vl are
at least 90, 95, 97, 98 or 99% identical to the respective parental
vh or vl, as long as the ABD is still able to bind to the target
antigen, as measured by at least one of a BIACORE.RTM., surface
plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g.
OCTET.RTM. assay) assay, with the latter finding particular use in
many embodiments.
L. Specific Bispecific Embodiments
[0606] The invention provides a number of particular bispecific
antibodies as outlined below.
[0607] 1. ICOS.times.PD-1
[0608] The invention provides bispecific heterodimeric antibodies
that bind ICOS and PD-1 each monovalently, and in some cases as
outlined herein, both bivalently.
[0609] In one embodiment, the PD-1 ABD is 1G6_L1.194_H1.279 and the
ICOS ABD is selected from sequences shown in FIG. 19, FIGS.
20A-20G, FIG. 24, FIG. 68A-68G and FIGS. 77A-77B as well as SEQ ID
NO: 27869-28086, 28087-28269, 27193-27335, 28549-28556 and
28557-28665, and those with the identifiers [ICOS]_H0.66_L0 and
[ICOS]_H0L0.
[0610] In one embodiment, the ICOS.times.PD-1 bispecific antibody
is selected from XENP numbers 20261, 20730, 20896, 22432-22438,
22731-22748, 22878-22894, 22931-22932, 22950-22961, 23090-23093,
23295-23296, 23301, 23405, 23408, 23410 (all without 428L/4345,
although they can have those substitutions); XENP numbers 22730,
22917-22928, 22935-22937, 22974-22979, 22995-22996, 23001, 23103,
23104 (all with 428L/4345, although they can not have those); XENP
numbers 23411, 21828, 21829, 21830, 21831, 22348, 23059 (using
prior art ICOS sequences); XENP numbers 18920, 24125, 24130
(additional bottle openers); XENP 23406, 23407, 24128
(ICOS.times.PD-1 central-scFv), XENP24123 (ICOS.times.PD-1
central-scFv2); XENP24134 (ICOS.times.PD-1 bispecific mAb) 24122
(ICOS.times.PD-1 DVD-Ig), XENP 24132, 24133 (ICOS.times.PD-1
Trident).
[0611] 2. ICOS.times.PD-L1
[0612] In this embodiment, the ICOS ABD is selected from sequences
shown in FIG. 19, FIGS. 20A-20G, FIG. 24, FIGS. 68A-68G and FIGS.
77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665, and those with the
identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0.
[0613] 3. ICOS.times.CTLA-4
[0614] In this embodiment, the ICOS ABD is selected from sequences
shown in FIG. 19, FIGS. 20A-20G, FIG. 24, FIGS. 68A-68G and FIGS.
77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665, and those with the
identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0.
[0615] In one embodiment, a bottle opener format with a Fab ICOS
ABD of [ICOS]H0.66_L0 is paired with a scFv CTLA-4 ABD of
[CTLA-4]_H3.32_L0.129, particularly in bottle opener backbone 1
from FIG. 9.
[0616] 4. ICOS.times.LAG-3
[0617] In this embodiment, the ICOS ABD is selected from sequences
shown in FIG. 19, FIGS. 20A-20G, FIG. 24, FIGS. 68A-68G and FIGS.
77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665, and those with the
identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0.
[0618] 5. ICOS.times.TIM-3
[0619] I this embodiment, the ICOS ABD is selected from sequences
shown in FIG. 19, FIGS. 20A-20G, FIG. 24, FIGS. 68A-68G and FIGS.
77A-77B as well as SEQ ID NO: 27869-28086, 28087-28269,
27193-27335, 28549-28556 and 28557-28665, and those with the
identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0.
M. Homologous Antibodies
[0620] The invention further provides antibodies that share amino
acid sequence identity with the antibodies outlined herein.
[0621] In one embodiment, bispecific antibodies are made that have
amino acid variants in one or more of the CDRs of the Vh and Vl
sequences outlined herein and in the Figures. In one embodiment,
antibodies are provided that have 1, 2, 3, 4 or 5 amino acid
differences in one or more of the CDRs of the vh and vl chains
outlined herein. These amino acid variants can be in one CDR or
spread out between more than one CDR. These amino acid variants can
also be in one or both of the Fvs of the bispecific antibody; e.g.
there can be 2 amino acid variants in CDRs on the ICOS Fv side
(generally a Fab but can be a scFv as outlined herein) and one on
the PD-1 side, etc.
[0622] Similarly, the invention provides for antibodies that have
at least 95, 96, 97 98 or 99% amino acid identity to the sequences
outlined herein, and particularly in the variable heavy and/or
variable light domains. This sequence identity can also be on one
Fv or both Fv of the bispecific antibodies. That is, bispecific
antibodies are provided that are 95-99% identical to the variable
heavy and/or variable light domains outlined in the figures.
IX. Nucleic Acids of the Invention
[0623] The invention further provides nucleic acid compositions
encoding the bispecific antibodies of the invention (or, in the
case of "monospecific" antibodies, nucleic acids encoding those as
well).
[0624] As will be appreciated by those in the art, the nucleic acid
compositions will depend on the format and scaffold of the
heterodimeric protein. Thus, for example, when the format requires
three amino acid sequences, three nucleic acid sequences can be
incorporated into one or more expression vectors for expression.
Similarly, some formats (e.g. dual scFv formats such as disclosed
in FIG. 2) only two nucleic acids are needed; again, they can be
put into one or two expression vectors. Some formats need 4 amino
acids (bispecific mAbs).
[0625] As is known in the art, the nucleic acids encoding the
components of the invention can be incorporated into expression
vectors as is known in the art, and depending on the host cells
used to produce the heterodimeric antibodies of the invention.
Generally, the nucleic acids are operably linked to any number of
regulatory elements (promoters, origin of replication, selectable
markers, ribosomal binding sites, inducers, etc.). The expression
vectors can be extra-chromosomal or integrating vectors.
[0626] The nucleic acids and/or expression vectors of the invention
are then transformed into any number of different types of host
cells as is well known in the art, including mammalian, bacterial,
yeast, insect and/or fungal cells, with mammalian cells (e.g. CHO
cells), finding use in many embodiments.
[0627] In some embodiments, nucleic acids encoding each monomer and
the optional nucleic acid encoding a light chain, as applicable
depending on the format, are each contained within a single
expression vector, generally under different or the same promoter
controls. In embodiments of particular use in the present
invention, each of these two or three nucleic acids are contained
on a different expression vector. As shown herein and in
62/025,931, hereby incorporated by reference, different vector
ratios can be used to drive heterodimer formation. That is,
surprisingly, while the proteins comprise first monomer:second
monomer:light chains (in the case of many of the embodiments herein
that have three polypeptides comprising the heterodimeric antibody)
in a 1:1:2 ratio, these are not the ratios that give the best
results.
[0628] The heterodimeric antibodies of the invention are made by
culturing host cells comprising the expression vector(s) as is well
known in the art. Once produced, traditional antibody purification
steps are done, including an ion exchange chromotography step. As
discussed herein, having the pIs of the two monomers differ by at
least 0.5 can allow separation by ion exchange chromatography or
isoelectric focusing, or other methods sensitive to isoelectric
point. That is, the inclusion of pI substitutions that alter the
isoelectric point (pI) of each monomer so that such that each
monomer has a different pI and the heterodimer also has a distinct
pI, thus facilitating isoelectric purification of the "triple F"
heterodimer (e.g., anionic exchange columns, cationic exchange
columns). These substitutions also aid in the determination and
monitoring of any contaminating dual scFv-Fc and mAb homodimers
post-purification (e.g., IEF gels, cIEF, and analytical IEX
columns).
X. Biological and Biochemical Functionality of the Heterodimeric
Immunomodulatory Antibodies
[0629] Generally the bispecific immunomodulatory antibodies of the
invention are administered to patients with cancer, and efficacy is
assessed, in a number of ways as described herein. Thus, while
standard assays of efficacy can be run, such as cancer load, size
of tumor, evaluation of presence or extent of metastasis, etc.,
immuno-oncology treatments can be assessed on the basis of immune
status evaluations as well. This can be done in a number of ways,
including both in vitro and in vivo assays. For example, evaluation
of changes in immune status (e.g. presence of ICOS+ CD4+ T cells
following ipi treatment) along with "old fashioned" measurements
such as tumor burden, size, invasiveness, LN involvement,
metastasis, etc. can be done. Thus, any or all of the following can
be evaluated: the inhibitory effects of PVRIG on CD4.sup.+ T cell
activation or proliferation, CD8.sup.+ T (CTL) cell activation or
proliferation, CD8.sup.+ T cell-mediated cytotoxic activity and/or
CTL mediated cell depletion, NK cell activity and NK mediated cell
depletion, the potentiating effects of PVRIG on Treg cell
differentiation and proliferation and Treg- or myeloid derived
suppressor cell (MDSC)-mediated immunosuppression or immune
tolerance, and/or the effects of PVRIG on proinflammatory cytokine
production by immune cells, e.g., IL-2, IFN-.gamma. or TNF-.alpha.
production by T or other immune cells.
[0630] In some embodiments, assessment of treatment is done by
evaluating immune cell proliferation, using for example, CFSE
dilution method, Ki67 intracellular staining of immune effector
cells, and 3H-Thymidine incorporation method,
[0631] In some embodiments, assessment of treatment is done by
evaluating the increase in gene expression or increased protein
levels of activation-associated markers, including one or more of:
CD25, CD69, CD137, ICOS, PD1, GITR, OX40, and cell degranulation
measured by surface expression of CD107A.
[0632] In general, gene expression assays are done as is known in
the art.
[0633] In general, protein expression measurements are also
similarly done as is known in the art.
[0634] In some embodiments, assessment of treatment is done by
assessing cytotoxic activity measured by target cell viability
detection via estimating numerous cell parameters such as enzyme
activity (including protease activity), cell membrane permeability,
cell adherence, ATP production, co-enzyme production, and
nucleotide uptake activity. Specific examples of these assays
include, but are not limited to, Trypan Blue or PI staining,
.sup.51Cr or .sup.35S release method, LDH activity, MTT and/or WST
assays, Calcein-AM assay, Luminescent based assay, and others.
[0635] In some embodiments, assessment of treatment is done by
assessing T cell activity measured by cytokine production, measure
either intracellularly in culture supernatant using cytokines
including, but not limited to, IFN.gamma., TNF.alpha., GM-CSF, IL2,
IL6, IL4, IL5, IL10, IL13 using well known techniques.
[0636] Accordingly, assessment of treatment can be done using
assays that evaluate one or more of the following: (i) increases in
immune response, (ii) increases in activation of .alpha..beta.
and/or .gamma..delta. T cells, (iii) increases in cytotoxic T cell
activity, (iv) increases in NK and/or NKT cell activity, (v)
alleviation of .alpha..beta. and/or .gamma..delta. T-cell
suppression, (vi) increases in pro-inflammatory cytokine secretion,
(vii) increases in IL-2 secretion; (viii) increases in
interferon-.gamma. production, (ix) increases in Th1 response, (x)
decreases in Th2 response, (xi) decreases or eliminates cell number
and/or activity of at least one of regulatory T cells (Tregs.
[0637] Assays to Measure Efficacy
[0638] In some embodiments, T cell activation is assessed using a
Mixed Lymphocyte Reaction (MLR) assay as is known in the art. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0639] In one embodiment, the signaling pathway assay measures
increases or decreases in immune response as measured for an
example by phosphorylation or de-phosphorylation of different
factors, or by measuring other post translational modifications. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0640] In one embodiment, the signaling pathway assay measures
increases or decreases in activation of .alpha..beta. and/or
.gamma..delta. T cells as measured for an example by cytokine
secretion or by proliferation or by changes in expression of
activation markers like for an example CD137, CD107a, PD1, etc. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0641] In one embodiment, the signaling pathway assay measures
increases or decreases in cytotoxic T cell activity as measured for
an example by direct killing of target cells like for an example
cancer cells or by cytokine secretion or by proliferation or by
changes in expression of activation markers like for an example
CD137, CD107a, PD1, etc. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0642] In one embodiment, the signaling pathway assay measures
increases or decreases in NK and/or NKT cell activity as measured
for an example by direct killing of target cells like for an
example cancer cells or by cytokine secretion or by changes in
expression of activation markers like for an example CD107a, etc.
An increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0643] In one embodiment, the signaling pathway assay measures
increases or decreases in .alpha..beta. and/or .gamma..delta.
T-cell suppression, as measured for an example by cytokine
secretion or by proliferation or by changes in expression of
activation markers like for an example CD137, CD107a, PD1, etc. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0644] In one embodiment, the signaling pathway assay measures
increases or decreases in pro-inflammatory cytokine secretion as
measured for example by ELISA or by Luminex or by Multiplex bead
based methods or by intracellular staining and FACS analysis or by
Alispot etc. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0645] In one embodiment, the signaling pathway assay measures
increases or decreases in IL-2 secretion as measured for example by
ELISA or by Luminex or by Multiplex bead based methods or by
intracellular staining and FACS analysis or by Alispot etc. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0646] In one embodiment, the signaling pathway assay measures
increases or decreases in interferon-.gamma. production as measured
for example by ELISA or by Luminex or by Multiplex bead based
methods or by intracellular staining and FACS analysis or by
Alispot etc. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0647] In one embodiment, the signaling pathway assay measures
increases or decreases in Th1 response as measured for an example
by cytokine secretion or by changes in expression of activation
markers. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0648] In one embodiment, the signaling pathway assay measures
increases or decreases in Th2 response as measured for an example
by cytokine secretion or by changes in expression of activation
markers. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0649] In one embodiment, the signaling pathway assay measures
increases or decreases cell number and/or activity of at least one
of regulatory T cells (Tregs), as measured for example by flow
cytometry or by IHC. A decrease in response indicates
immunostimulatory activity. Appropriate decreases are the same as
for increases, outlined below.
[0650] In one embodiment, the signaling pathway assay measures
increases or decreases in M2 macrophages cell numbers, as measured
for example by flow cytometry or by IHC. A decrease in response
indicates immunostimulatory activity. Appropriate decreases are the
same as for increases, outlined below.
[0651] In one embodiment, the signaling pathway assay measures
increases or decreases in M2 macrophage pro-tumorigenic activity,
as measured for an example by cytokine secretion or by changes in
expression of activation markers. A decrease in response indicates
immunostimulatory activity. Appropriate decreases are the same as
for increases, outlined below.
[0652] In one embodiment, the signaling pathway assay measures
increases or decreases in N2 neutrophils increase, as measured for
example by flow cytometry or by IHC. A decrease in response
indicates immunostimulatory activity. Appropriate decreases are the
same as for increases, outlined below.
[0653] In one embodiment, the signaling pathway assay measures
increases or decreases in N2 neutrophils pro-tumorigenic activity,
as measured for an example by cytokine secretion or by changes in
expression of activation markers. A decrease in response indicates
immunostimulatory activity. Appropriate decreases are the same as
for increases, outlined below.
[0654] In one embodiment, the signaling pathway assay measures
increases or decreases in inhibition of T cell activation, as
measured for an example by cytokine secretion or by proliferation
or by changes in expression of activation markers like for an
example CD137, CD107a, PD1, etc. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0655] In one embodiment, the signaling pathway assay measures
increases or decreases in inhibition of CTL activation as measured
for an example by direct killing of target cells like for an
example cancer cells or by cytokine secretion or by proliferation
or by changes in expression of activation markers like for an
example CD137, CD107a, PD1, etc. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0656] In one embodiment, the signaling pathway assay measures
increases or decreases in .alpha..beta. and/or .gamma..delta. T
cell exhaustion as measured for an example by changes in expression
of activation markers. A decrease in response indicates
immunostimulatory activity. Appropriate decreases are the same as
for increases, outlined below.
[0657] In one embodiment, the signaling pathway assay measures
increases or decreases .alpha..beta. and/or .gamma..delta. T cell
response as measured for an example by cytokine secretion or by
proliferation or by changes in expression of activation markers
like for an example CD137, CD107a, PD1, etc. An increase in
activity indicates immunostimulatory activity. Appropriate
increases in activity are outlined below.
[0658] In one embodiment, the signaling pathway assay measures
increases or decreases in stimulation of antigen-specific memory
responses as measured for an example by cytokine secretion or by
proliferation or by changes in expression of activation markers
like for an example CD45RA, CCR7 etc. An increase in activity
indicates immunostimulatory activity. Appropriate increases in
activity are outlined below.
[0659] In one embodiment, the signaling pathway assay measures
increases or decreases in apoptosis or lysis of cancer cells as
measured for an example by cytotoxicity assays such as for an
example MTT, Cr release, Calcine AM, or by flow cytometry based
assays like for an example CFSE dilution or propidium iodide
staining etc. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0660] In one embodiment, the signaling pathway assay measures
increases or decreases in stimulation of cytotoxic or cytostatic
effect on cancer cells. as measured for an example by cytotoxicity
assays such as for an example MTT, Cr release, Calcine AM, or by
flow cytometry based assays like for an example CFSE dilution or
propidium iodide staining etc. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0661] In one embodiment, the signaling pathway assay measures
increases or decreases direct killing of cancer cells as measured
for an example by cytotoxicity assays such as for an example MTT,
Cr release, Calcine AM, or by flow cytometry based assays like for
an example CFSE dilution or propidium iodide staining etc. An
increase in activity indicates immunostimulatory activity.
Appropriate increases in activity are outlined below.
[0662] In one embodiment, the signaling pathway assay measures
increases or decreases Th17 activity as measured for an example by
cytokine secretion or by proliferation or by changes in expression
of activation markers. An increase in activity indicates
immunostimulatory activity. Appropriate increases in activity are
outlined below.
[0663] In one embodiment, the signaling pathway assay measures
increases or decreases in induction of complement dependent
cytotoxicity and/or antibody dependent cell-mediated cytotoxicity,
as measured for an example by cytotoxicity assays such as for an
example MTT, Cr release, Calcine AM, or by flow cytometry based
assays like for an example CFSE dilution or propidium iodide
staining etc. An increase in activity indicates immunostimulatory
activity. Appropriate increases in activity are outlined below.
[0664] In one embodiment, T cell activation is measured for an
example by direct killing of target cells like for an example
cancer cells or by cytokine secretion or by proliferation or by
changes in expression of activation markers like for an example
CD137, CD107a, PD1, etc. For T-cells, increases in proliferation,
cell surface markers of activation (e.g. CD25, CD69, CD137, PD1),
cytotoxicity (ability to kill target cells), and cytokine
production (e.g. IL-2, IL-4, IL-6, IFN.gamma., TNF-a, IL-10,
IL-17A) would be indicative of immune modulation that would be
consistent with enhanced killing of cancer cells.
[0665] In one embodiment, NK cell activation is measured for
example by direct killing of target cells like for an example
cancer cells or by cytokine secretion or by changes in expression
of activation markers like for an example CD107a, etc. For NK
cells, increases in proliferation, cytotoxicity (ability to kill
target cells and increases CD107a, granzyme, and perforin
expression), cytokine production (e.g. IFN.gamma. and TNF), and
cell surface receptor expression (e.g. CD25) would be indicative of
immune modulation that would be consistent with enhanced killing of
cancer cells.
[0666] In one embodiment, .gamma..delta. T cell activation is
measured for example by cytokine secretion or by proliferation or
by changes in expression of activation markers.
[0667] In one embodiment, Th1 cell activation is measured for
example by cytokine secretion or by changes in expression of
activation markers.
[0668] Appropriate increases in activity or response (or decreases,
as appropriate as outlined above), are increases of 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the
signal in either a reference sample or in control samples, for
example test samples that do not contain an anti-PVRIG antibody of
the invention. Similarly, increases of at least one-, two-, three-,
four- or five-fold as compared to reference or control samples show
efficacy.
XI. Treatments
[0669] Once made, the compositions of the invention find use in a
number of oncology applications, by treating cancer, generally by
inhibiting the suppression of T cell activation (e.g. T cells are
no longer suppressed) with the binding of the bispecific
immunomodulatory antibodies of the invention.
[0670] Accordingly, the heterodimeric compositions of the invention
find use in the treatment of these cancers.
XII. Antibody Compositions for In Vivo Administration
[0671] Formulations of the antibodies used in accordance with the
present invention are prepared for storage by mixing an antibody
having the desired degree of purity with optional pharmaceutically
acceptable carriers, excipients or stabilizers (as generally
outlined in Remington's Pharmaceutical Sciences 16th edition, Osol,
A. Ed. [1980]), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, buffers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
Administrative Modalities
[0672] The antibodies and chemotherapeutic agents of the invention
are administered to a subject, in accord with known methods, such
as intravenous administration as a bolus or by continuous infusion
over a period of time.
Treatment Modalities
[0673] In the methods of the invention, therapy is used to provide
a positive therapeutic response with respect to a disease or
condition. By "positive therapeutic response" is intended an
improvement in the disease or condition, and/or an improvement in
the symptoms associated with the disease or condition. For example,
a positive therapeutic response would refer to one or more of the
following improvements in the disease: (1) a reduction in the
number of neoplastic cells; (2) an increase in neoplastic cell
death; (3) inhibition of neoplastic cell survival; (5) inhibition
(i.e., slowing to some extent, preferably halting) of tumor growth;
(6) an increased patient survival rate; and (7) some relief from
one or more symptoms associated with the disease or condition.
[0674] Positive therapeutic responses in any given disease or
condition can be determined by standardized response criteria
specific to that disease or condition. Tumor response can be
assessed for changes in tumor morphology (i.e., overall tumor
burden, tumor size, and the like) using screening techniques such
as magnetic resonance imaging (MRI) scan, x-radiographic imaging,
computed tomographic (CT) scan, bone scan imaging, endoscopy, and
tumor biopsy sampling including bone marrow aspiration (BMA) and
counting of tumor cells in the circulation.
[0675] In addition to these positive therapeutic responses, the
subject undergoing therapy may experience the beneficial effect of
an improvement in the symptoms associated with the disease.
[0676] Treatment according to the present invention includes a
"therapeutically effective amount" of the medicaments used. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve a desired
therapeutic result.
[0677] A therapeutically effective amount may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the medicaments to elicit a desired
response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody
or antibody portion are outweighed by the therapeutically
beneficial effects.
[0678] A "therapeutically effective amount" for tumor therapy may
also be measured by its ability to stabilize the progression of
disease. The ability of a compound to inhibit cancer may be
evaluated in an animal model system predictive of efficacy in human
tumors.
[0679] Alternatively, this property of a composition may be
evaluated by examining the ability of the compound to inhibit cell
growth or to induce apoptosis by in vitro assays known to the
skilled practitioner. A therapeutically effective amount of a
therapeutic compound may decrease tumor size, or otherwise
ameliorate symptoms in a subject. One of ordinary skill in the art
would be able to determine such amounts based on such factors as
the subject's size, the severity of the subject's symptoms, and the
particular composition or route of administration selected.
[0680] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. Parenteral compositions may be formulated in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited
as unitary dosages for the subjects to be treated; each unit
contains a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0681] The specification for the dosage unit forms of the present
invention are dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0682] The efficient dosages and the dosage regimens for the
bispecific antibodies used in the present invention depend on the
disease or condition to be treated and may be determined by the
persons skilled in the art.
[0683] An exemplary, non-limiting range for a therapeutically
effective amount of an bispecific antibody used in the present
invention is about 0.1-100 mg/kg.
[0684] All cited references are herein expressly incorporated by
reference in their entirety.
[0685] Whereas particular embodiments of the invention have been
described above for purposes of illustration, it will be
appreciated by those skilled in the art that numerous variations of
the details may be made without departing from the invention as
described in the appended claims.
XIII. Examples
[0686] Examples are provided below to illustrate the present
invention. These examples are not meant to constrain the present
invention to any particular application or theory of operation. For
all constant region positions discussed in the present invention,
numbering is according to the EU index as in Kabat (Kabat et al.,
1991, Sequences of Proteins of Immunological Interest, 5th Ed.,
United States Public Health Service, National Institutes of Health,
Bethesda, entirely incorporated by reference). Those skilled in the
art of antibodies will appreciate that this convention consists of
nonsequential numbering in specific regions of an immunoglobulin
sequence, enabling a normalized reference to conserved positions in
immunoglobulin families. Accordingly, the positions of any given
immunoglobulin as defined by the EU index will not necessarily
correspond to its sequential sequence.
[0687] General and specific scientific techniques are outlined in
US Publications 2015/0307629, 2014/0288275 and WO2014/145806, all
of which are expressly incorporated by reference in their entirety
and particularly for the techniques outlined therein.
A. Example 1: TILs from Multiple Cancer Types Co-Express PD-1 and T
Cell Costimulatory Receptors
[0688] To investigate potential associations between PD-1 and
various T cell costimulatory receptors, RNA sequencing data from
The Cancer Genome Atlas project (TCGA) were used for analysis. V2
RSEM data were downloaded from FireBrowse (http://firebrowse.org/).
Analysis was performed using R with custom routines. The
correlation between the expression of PD-1 and eight costimulatory
receptors is depicted in FIG. 1, along with calculated R2 values
(square of the Pearson correlation coefficient). The data show that
PD-1 and several costimulatory receptors were co-expressed in
cancers including bladder, breast, colon, head & neck, kidney,
lung-adeno, lung squamous, ovarian, pancreatic, prostate, and
melanoma cancer. Notably, expression of ICOS on TILs correlates
better with that of PD-1 than several other costims.
B. Example 2: Immune Checkpoint Antigen Binding Domains
[0689] 1. 2A: Anti-PD-1 ABDs
[0690] Examples of antibodies which bind PD-1 were generated in
bivalent IgG1 format with E233P/L234V/L235A/G236del/S267K
substitutions, illustrative sequences for which are depicted in
FIG. 10. DNA encoding the variable regions was generated by gene
synthesis and was inserted into the mammalian expression vector
pTT5 by the Gibson Assembly method. Heavy chain VH genes were
inserted via Gibson Assembly into pTT5 encoding the human IgG1
constant region with the substitutions mentioned above. Light
chains VL genes were inserted into pTT5 encoding the human C?
constant region. DNA was transfected into HEK293E cells for
expression. Additional PD-1 ABDs (including those derived from the
above antibodies) were formatted as Fabs and scFvs for use in
costim/checkpoint bispecific antibodies, illustrative sequences for
which are depicted respectively in FIG. 11 and in the sequence
listing.
[0691] 2. 2B: Anti-CTLA-4 ABDs
[0692] Antibodies which bind CTLA-4 were generated in bivalent IgG1
format with E233P/L234V/L235A/G236del/S267K substitutions. DNA
encoding the variable regions was generated by gene synthesis and
was inserted into the mammalian expression vector pTT5 by the
Gibson Assembly method. Heavy chain VH genes were inserted via
Gibson Assembly into pTT5 encoding the human IgG1 constant region
with the substitutions mentioned above. Light chains VL genes were
inserted into pTT5 encoding the human C? constant region. DNA was
transfected into HEK293E cells for expression. Additional CTLA-4
ABDs (including those derived from the above antibodies) were
formatted as scFvs for use in costim/checkpoint bispecific
antibodies, illustrative sequences for which are depicted in FIG.
12 and in the sequence listing.
[0693] 3. 2C: Anti-LAG-3 ABDs
[0694] Examples of antibodies which bind LAG-3 were generated in
bivalent IgG1 format with E233P/L234V/L235A/G236del/S267K
substitutions. DNA encoding the variable regions was generated by
gene synthesis and was inserted into the mammalian expression
vector pTT5 by the Gibson Assembly method. Heavy chain VH genes
were inserted via Gibson Assembly into pTT5 encoding the human IgG1
constant region with the substitutions mentioned above. Light
chains VL genes were inserted into pTT5 encoding the human C?
constant region. DNA was transfected into HEK293E cells for
expression. Additional LAG-3 ABDs (including those derived from the
above antibodies) were formatted as Fabs for use in
costim/checkpoint bispecific antibodies, illustrative sequences for
which are depicted in FIG. 13 and in the sequence listing.
[0695] 4. 2D: Anti-TIM-3 ABDs
[0696] Examples of antibodies which bind TIM-3 were generated in
bivalent IgG1 format with E233P/L234V/L235A/G236del/S267K
substitutions, exemplary sequences for which are depicted in FIG.
14. DNA encoding the variable regions was generated by gene
synthesis and was inserted into the mammalian expression vector
pTT5 by the Gibson Assembly method. Heavy chain VH genes were
inserted via GIBSON ASSEMBLY.RTM. into pTT5 encoding the human IgG1
constant region with the substitutions mentioned above. Light
chains V.sub.L genes were inserted into pTT5 encoding the human C?
constant region. DNA was transfected into HEK293E cells for
expression. The above antibodies were formatted as Fabs for use in
costim/checkpoint bispecific antibodies.
[0697] 5. 2E: Anti-PD-L1 ABDs
[0698] Prototype antibodies which bind PD-L1 were generated in
bivalent IgG1 format with E233P/L234V/L235A/G236del/S267K
substitutions, exemplary sequences for which are depicted in FIG.
15A-15C. DNA encoding the variable regions was generated by gene
synthesis and was inserted into the mammalian expression vector
pTT5 by the Gibson Assembly method. Heavy chain VH genes were
inserted via GIBSON ASSEMBLY.RTM. into pTT5 encoding the human IgG1
constant region with the substitutions mentioned above. Light
chains VL genes were inserted into pTT5 encoding the human C?
constant region. DNA was transfected into HEK293E cells for
expression. The above antibodies were formatted as Fabs and scFvs
for use in costim/checkpoint bispecific antibodies.
C. Example 3: Costimulatory Receptor Antigen Binding Domains
[0699] Prototype costimulatory receptor antibodies which bind ICOS,
GITR, OX40, and 4-1BB were generated in bivalent IgG1 format with
E233P/L234V/L235A/G236del/S267K substitutions, sequences for which
are depicted in FIGS. 16-19. DNA encoding the variable regions was
generated by gene synthesis and was inserted into the mammalian
expression vector pTT5 by the Gibson Assembly method. Heavy chain
VH genes were inserted via GIBSON ASSEMBLY.RTM. into pTT5 encoding
the human IgG1 constant region with the substitutions mentioned
above. Light chains VL genes were inserted into pTT5 encoding the
human C? constant region. DNA was transfected into HEK293E cells
for expression. The above antibodies were formatted as Fabs or
scFvs for use in costim/checkpoint bispecific antibodies.
D. Example 4: Engineering Anti-ICOS ABD for Stability and
Affinity
[0700] The parental variable region of an anti-ICOS antibody
(XENP16435; depicted in FIG. 19) was engineered for use as a
component in an ICOS.times.checkpoint bispecific antibody. A
library of Fv variants engineered to have optimal affinity and
stability was constructed by site-directed mutagenesis
(QUIKCHANGE.RTM. Stratagene, Cedar Creek, Tx.) or additional gene
synthesis and subcloning in Fab-His and scFv-His formats and
produced as described below.
[0701] 1. 4A: Anti-ICOS Fabs
[0702] Amino acid sequences for variant anti-ICOS Fabs are listed
in FIGS. 20A-20G (the polyhistidine (His6) tags have been removed
from the C-terminal of the Fab heavy chains). DNA encoding the two
chains needed for Fab expression were generated by gene synthesis
and were subcloned using standard molecular biology techniques into
the expression vector pTT5. The Fab heavy chain included a
C-terminal polyhistidine tag. DNA was transfected into HEK293E
cells for expression and resulting proteins were purified from the
supernatant using Ni-NTA chromatography. The resultant anti-ICOS
Fabs were characterized for stability and affinity.
[0703] Differential Scanning Fluorimetry (DSF) experiments were
performed using a Bio-Rad CFX CONNECT.TM. Real-Time PCR Detection
System. Proteins were mixed with SYPRO Orange fluorescent dye and
diluted to 0.2 mg/mL in PBS. The final concentration of SYPRO
Orange was 10.times.. After an initial 10 minute incubation period
at 25.degree. C., proteins were heated from 25 to 95.degree. C.
using a heating rate of 1.degree. C./min. A fluorescence
measurement was taken every 30 seconds. Melting temperatures (Tm)
were calculated using the instrument software. The results are
shown in FIG. 21.
[0704] A series of affinity screens of the anti-ICOS Fabs to human
ICOS were performed using OCTET.RTM., a BioLayer Interferometry
(BLI)-based method. Experimental steps for OCTET.RTM. generally
included the following: Immobilization (capture of ligand or test
article onto a biosensor); Association (dipping of ligand- or test
article-coated biosensors into wells containing serial dilutions of
the corresponding test article or ligand); and Dissociation
(returning of biosensors to well containing buffer) in order to
determine the monovalent affinity of the test articles.
Specifically, anti-mouse Fc (AMC) biosensors were used to capture
mouse IgG2a Fc fusion of ICOS and dipped into multiple
concentrations of the test articles. The resulting equilibrium
dissociation constants (KD), association rates (ka), and
dissociation rates (kd) are presented in FIGS. 22-23. Binding
affinities and kinetic rate constants were obtained by analyzing
the processed data using a 1:1 binding model using ForteBio
OCTET.RTM. Data Analysis software (ForteBio). The data from a two
separate experiments are depicted in FIG. 22A-22C. In a further
experiment, streptavidin (SA) biosensors were used to capture
ICOS-TEV-Fc-Avi and dipped into the test articles. The data are
depicted in FIG. 23. A number of variant anti-ICOS Fabs including
XENP22780, XENP22782, XENP22783, and XENP22784 had improved
stability while maintaining affinity characteristics similar to a
Fab comprising the parental variable regions (XENP22050).
[0705] 2. 4B: Anti-ICOS scFvs
[0706] Amino acid sequences for anti-ICOS scFvs are listed in FIG.
24 (the polyhistidine (His6) tags have been removed from the
C-terminal of the scFvs). DNA encoding the scFv was generated by
gene synthesis and were subcloned using standard molecular biology
techniques into the expression vector pTT5. The scFv included a
C-terminal polyhistidine tag. DNA was transfected into HEK293E
cells for expression and resulting proteins were purified from the
supernatant using Ni-NTA chromatography. The resultant anti-ICOS
scFvs were characterized for stability in DSF experiments as
described above. The data is depicted in FIG. 25.
E. Example 5: Anti-ICOS.times.Anti-PD-1 Bispecific Antibodies
[0707] 1. 5A: Prototype Costim/Checkpoint Bottle-Openers
[0708] Schematics for costim/checkpoint bispecific antibody in the
bottle-opener format are depicted as FIG. 2A. Prototype
bottle-openers with costimulatory receptor binding Fab arms based
on prototype anti-GITR mAb (XENP16438), anti-OX40 mAb (XENP16437),
anti-4-1BB mAb (XENP14410) and anti-ICOS mAb (XENP16435) and
exemplary anti-PD-1 scFv (XENP19692) arm were produced to
investigate their effect on cytokine secretion in an SEB-stimulated
PBMC assay. Sequences are depicted in FIG. 26A-26D. DNA encoding
the three chains needed for bottle-opener expression were generated
by gene synthesis and were subcloned using standard molecular
biology techniques into the expression vector pTT5. DNA transfected
into HEK293E cells for expression and resulting proteins were
purified using standard techniques.
[0709] a. 5A(a): Prototype Anti-ICOS.times.Anti-PD-1 Bottle-Opener
Enhances Cytokine Secretion
[0710] Staphylococcal Enterotoxin B (SEB) is a superantigen that
causes T cell activation and proliferation in a manner similar to
that achieved by activation via the T cell receptor (TCR).
Stimulating human PBMC with SEB is a common method for assaying T
cell activation and proliferation. PBMCs were simulated with 100
ng/mL SEB for 2 days. Cells were washed twice and restimulated with
100 ng/mL SEB in combination with 20 .mu.g/mL of the indicated test
articles. A first bivalent anti-PD-1 antibody based on nivolumab
(XENP16432), a second bivalent anti-PD-1 mAb (XENP19686), and a
bivalent anti-RSV mAb (XENP15074) were used as controls. 24 hours
after treatment, supernatants were assayed for IL-2. The data
depicted in FIG. 27 show that each of the costim/checkpoint
bottle-openers enhanced cytokine secretion in comparison to the
bivalent anti-RSV antibody. Surprisingly, induction of cytokine
secretion by XENP22730 is vastly superior to cytokine secretion by
bivalent anti-PD-1 antibody alone as well as the other prototype
costim/checkpoint bottle-openers, indicating that addition of ICOS
binding enhances cytokine production and that ICOS is a better PD-1
partner than other costimulatory receptors for a bispecific
antibody.
[0711] In another experiment, PBMCs were stimulated with 0.01
.mu.g/mL SEB for 3 days with 20 .mu.g/mL of indicated test
articles. As control, test articles were also incubated with naive
(non-SEB stimulated) PBMCs. 3 days after treatment, supernatant was
assessed for IL-2 secretion as an indicator of T cell activation
(depicted in FIG. 28). The data show that neither the anti-PD-1
bivalent antibody nor the anti-ICOS.times.anti-PD-1 bottle-opener
stimulate IL-2 secretion in naive cells. Further, the data show
again that XENP20896 enhances IL-2 secretion more than the bivalent
anti-PD-1 antibody alone does and that XENP20896 enhances IL-2
secretion more than combination of bivalents (XENP16432 and
XENP16435) as well as combination of one-arms (XENP20111 and
XENP20266) do.
[0712] Costimulatory receptors such as ICOS have previously been
found to induce cytokine production only following crosslinking by
bivalent antibodies or multimerized ligands (Viera et al. 2004;
Sanmamed et al. 2015). In view of the crosslinking mechanisms, it
is surprising that monovalent ICOS binding by a single arm in a
costim.times.checkpoint blockade bispecific antibody was able to
enhance cytokine production. Further, it is notable that the
bispecific antibody was able to enhance cytokine secretion more
than bivalent anti-PD-1 mAb in combination with bivalent anti-ICOS
mAb (XENP16432+XENP16435).
[0713] b. 5A(b): PD-1 and ICOS Double-Positive Cells are
Selectively Occupied by Prototype Anti-ICOS.times.Anti-PD-1
Bottle-Opener
[0714] Selective targeting of tumor-reactive TILs co-expressing
immune checkpoint receptors (e.g. PD-1) and costimulatory receptors
as shown in Example 1 over non-tumor reactive T cells expressing
immune checkpoint receptors or costimulatory receptors alone could
enhance anti-tumor activity while avoiding peripheral toxicity (as
depicted in FIG. 29).
[0715] An SEB-stimulated PBMC assay was used to investigate binding
of anti-ICOS.times.anti-PD-1 bottle-opener to T cells. PBMCs were
stimulated with 100 ng/mL SEB (staphylococcal enterotoxin B) for 3
days, after which the PBMCs were treated with the indicated test
articles for 30 minutes at 4.degree. C. PBMCs were then incubated
with APC-labeled one-arm anti-ICOS antibody and FITC-labeled
one-arm anti-PD-1 antibody for 30 minutes at 4.degree. C. FIGS. 30
and 31 shows receptor occupancy of a prototype
anti-ICOS.times.anti-PD-1 bottle-opener (XENP20896), one-arm
anti-ICOS antibody (XENP20266) and one-arm anti-PD-1 antibody
(XENP20111).
[0716] The data show that double-positive cells (expressing both
PD-1 and ICOS) are selectively occupied by the
anti-ICOS.times.anti-PD-1 bottle-opener (XENP20896) as depicted in
FIG. 30, indicating that monovalent ICOS and PD-1 binding is useful
for selective targeting. Further, anti-ICOS.times.anti-PD-1
bottle-opener (e.g. XENP20896) binds more potently to
double-positive cells than monovalent, monospecific one-arm
anti-PD-1 and anti-ICOS as shown in FIG. 31A.
[0717] c. 5A(c): Prototype Anti-ICOS.times.Anti-PD-1 Bottle-Opener
Enhance Engraftment in a GVHD Mouse Study
[0718] The prototype anti-ICOS.times.anti-PD-1 bottle-opener was
evaluated in a Graft-versus-Host Disease (GVHD) model conducted in
NSG (NOD-SCID-gamma) immunodeficient mice. The mice were engrafted
with human PBMCs. When NSG mice are injected with human PBMCs, they
develop an autoimmune response against the human PBMCs. Treatment
of NSG mice injected with human PBMCs with immune checkpoint
antibodies (e.g. anti-PD-1) enhance engraftment. Thus, increased
engraftment shows efficacy of the antibodies.
[0719] 10 million human PBMCs were engrafted into NSG mice via
IV-OSP on Day 0 followed by dosing with the indicated test articles
on Day 1, 8, 15, and 22. Human CD45+ cell counts were measured on
Day 14 as an indicator of disease.
[0720] The data depicted in Figure A show that the
anti-ICOS.times.anti-PD-1 bottle-opener enhance proliferation of
CD45+ cells in human PBMC-engrafted NSG mice as compared to
controls (PBS and PBS+PBMC). Further, enhancement is greater using
antibodies of the invention than that seen with bivalent anti-PD-1
antibody.
[0721] 2. 5B: Production of Variant Anti-ICOS.times.Anti-PD-1
Bottle-Openers with Optimized Anti-ICOS Fab Arms
[0722] Variant anti-ICOS.times.anti-PD-1 bottle-openers comprising
anti-ICOS Fabs engineered as described in Example 4A were produced
as generally described above. Amino acid sequences for variant
anti-ICOS.times.anti-PD-1 bottle-openers are listed in FIG. 32.
Amino acid sequences for variant anti-ICOS.times.anti-PD-1 with
FcRn pH 6.0 affinity enhancing substitutions are listed in FIG.
33.
[0723] The resultant anti-ICOS.times.anti-PD-1 bispecific
antibodies were characterized for affinity to human and cynomolgus
ICOS using Octet as generally described above. In a first set of
experiments, AMC biosensors were used to capture mouse IgG2a Fc
fusion of ICOS and dipped into multiple concentrations of the test
articles (data depicted in FIG. 34A-3). In further experiments,
streptavidin (SA or SAX) biosensors were used to capture
biotinylated human and cynomolgus IgG1 Fc fusions of human and
cynomolgus ICOS and dipped into multiple concentrations of the test
articles (data depicted in FIG. 35).
[0724] 3. 5C:T Cell Surface Binding of Variant
Anti-ICOS.times.Anti-PD-1 Bottle-Openers
[0725] Binding of anti-ICOS.times.anti-PD-1 bispecifics to T cells
was measured in an SEB-stimulated PBMC assay. Human PBMCs were
stimulated with 100 ng/mL SEB for 3 days. PBMCs were then treated
with the indicated test articles and incubated at 4.degree. C. for
30 minutes. After treatment, cells were incubated with FITC-labeled
anti-CD3 antibody and APC-labeled anti-human IgG Fc secondary
antibody. MFI on CD3+ cells are depicted in FIG. 36A-36B.
[0726] 4. 5D: Receptor Occupancy of Variant
Anti-ICOS.times.Anti-PD-1 Bottle-Openers on T Cells
[0727] Receptor occupancy of variant anti-ICOS.times.anti-PD-1
bottle-openers on T cells was measured in an SEB-stimulated PBMC
assay. PBMCs were stimulated with 100 ng/mL SEB (staphylococcal
enterotoxin B) for 3 days, after which the PBMCs were treated with
the indicated test articles for 30 minutes at 4.degree. C. PBMCs
were then incubated with APC-labeled one-arm anti-ICOS antibody and
FITC-labeled one-arm anti-PD-1 antibody for 30 minutes at 4.degree.
C. FIG. 37 depicts the receptor occupancy of variant
anti-ICOS.times.anti-PD-1 bispecific antibodies, corresponding
one-arm anti-ICOS antibodies and one-arm anti-PD-1 antibody
(XENP20111) on PD-1 and ICOS double-positive T cells. Consistent
with the prototype antibodies investigated in Example 1A, each of
the bottle-openers binds more potently to double-positive cells
than monovalent, monospecific one-arm anti-PD-1 and one-arm
anti-ICOS antibodies.
[0728] 5. 5E: In Vitro Activity of Variant
Anti-ICOS.times.Anti-PD-1 Bottle-Openers in a Cytokine Release
Assay
[0729] Human PBMCs were stimulated with 100 ng/mL SEB for 2 days.
Cells were washed and stimulated again with 100 ng/mL SEB in
combination with 20 .mu.g/mL of indicated test articles. 24 hours
after treatment, cells were assayed for IL-2 (FIG. 38A) and IFN?
(FIG. 38B). The data show that anti-ICOS.times.anti-PD-1
bottle-openers stimulated significantly more cytokine release than
bivalent anti-PD-1 antibody (XENP16432) alone, bivalent anti-ICOS
antibody (XENP16435) alone, or bivalent anti-PD-1 antibody plus
bivalent anti-ICOS antibody in combination.
[0730] In a further experiment, human PBMCs were stimulated with
100 ng/mL SEB for 2 days. Cells were washed and stimulated again
with 100 ng/mL SEB in combination with 20 .mu.g/mL of indicated
test articles. 24 hours after treatment, cells were assayed for
IL-2 (FIG. 39A) and IFN? (FIG. 39B).
[0731] 6. 5F: In Vivo Activity of Variant Anti-ICOS.times.Anti-PD-1
Bottle Openers in a GVHD Mouse Study
[0732] In a first study, 10 million human PBMCs were engrafted into
NSG mice via IV-OSP on Day 0 followed by dosing with the indicated
test articles on Day 1. IFN? levels and human CD45+, CD8+ T cell
and CD4+ T cell counts were measured on Day 7, 11 and 14. FIGS.
40A-B respectively depicts IFN? levels on Day 7 and 11. FIGS. 41A-B
respectively depict CD45+ cell counts on Day 11 and 14. FIGS. 42A-B
respectively depict CD8+ T cell and CD4+ T cell counts on Day 14.
FIG. 43 depicts the change in body weight in the mice by Day 14
resulting from exacerbation of GVHD due to T cell expansion and
IFN? production.
[0733] In a further study with additional variant
anti-ICOS.times.anti-PD-1 bottle-openers, 10 million human PBMCs
were engrafted into NSG mice via IV-OSP on Day 0 followed by dosing
with the indicated test articles at indicated concentrations on Day
1. IFN.gamma. levels and human CD45+, CD8+ T cell and CD4+ T cell
counts were measured on Day 7 and 14. FIG. 44A-44B respectively
depicts IFN? levels on Day 7 and 14. FIG. 45 depicts CD45+ cell
count on Day 14. FIG. 46A-C respectively depict CD8+ T cell count,
CD4+ T cell counts and CD8+/CD4+ ratio on Day 14. Body weight of
mice were also measured on Day 12 and 15 and depicted respectively
in FIG. 47A-B as percentage of initial body weight.
[0734] The Figures show that the anti-ICOS.times.anti-PD-1
bottle-openers enhance engraftment (as indicated by the
proliferation of CD45+ cells, CD8+ T cells and CD4+ T cells and
decrease in body weight of mice). The observed activity is
correlated to the in vitro potency of each variant. Further, a
number of the bottle-openers including XENP20896, XENP22744,
XENP23092, XENP22730, XENP23104, XENP22974, and XENP23411 enhance
engraftment much more than the control bivalent anti-PD-1 antibody
(XENP16432) does.
[0735] 7. 5G: Additional Anti-ICOS ABDs Work in
Anti-ICOS.times.Anti-PD-1 Bottle Opener
[0736] Additional anti-ICOS.times.anti-PD-1 bottle-openers were
produced comprising anti-ICOS Fabs based on other anti-ICOS ABDs
described in Example 3 as generally described above. Sequences for
the additional bottle-openers are depicted in FIG. 48A-48D.
[0737] In a first experiment, PBMCs were stimulated with 100 ng/mL
SEB for 2 days. Cells were washed twice and restimulated with 100
ng/mL SEB in combination with 20 .mu.g/mL of indicated test
articles (PBS as control). 24 hours after treatment, supernatants
were assayed for IL-2 concentration as depicted in FIG. 49. In a
second experiment, PBMCs were stimulated with 10 ng/mL SEB and
treated with 20 .mu.g/mL of indicated test articles for 3 days
(bivalent anti-PD-1 XENP16432 as control). Supernatants were
collected and assays for IL-2. FIG. 50 depicts the fold induction
in IL-2 over bivalent anti-RSV mAb.
[0738] Consistent with the data in Example 5A(a), XENP20896
stimulated secretion of IL-2. Notably, the additional
bottle-openers comprising alternative anti-ICOS ABDs were also able
to stimulate secretion of IL-2 demonstrating that the enhancement
of cytokine secretion by an anti-ICOS.times.anti-PD-1 bottle-opener
is not unique to ABDs derived from the parental anti-ICOS ABD
described in Example 4.
[0739] 8. 5H: Clear ICOS Signature is Exhibited by
Anti-ICOS.times.Anti-PD-1 Bottle-Openers
[0740] a. 5H(a): Anti-ICOS.times.Anti-PD-1 Bispecific Antibodies
Induce AKT Phosphorylation
[0741] ICOS ligation induces AKT phosphorylation in activated T
cells (Fos, C et al. 2008), and as such, AKT phosphorylation would
be an indicator of ICOS agonism by anti-ICOS.times.anti-PD-1
bispecific antibodies of the invention.
[0742] Human PBMCs were stimulated with 100 ng/mL SEB for 2 days.
Following stimulation, CD3+ cells were isolated by negative
selection using EasySep.TM. Human T Cell Enrichment Kit (STEMCELL
Technologies, Vancouver, Canada) and then treated with indicated
test articles in combination with plate bound anti-CD3 antibody
(OKT3; 500 ng/mL). Cells were lysed 30 minutes after treatment and
assayed for total AKT and phosphorylated AKT (Ser473) by a
multiplexed phosphoprotein assay on MULTI-SPOT 384-Well Spot plates
(Meso Scale Discovery, Rockville, Md.). The data are depicted in
FIG. 51 as percentage of AKT phosphorylated following
treatment.
[0743] The data shows no increase in AKT phosphorylation following
treatment with negative control bivalent anti-PD-1 mAb (XENP16432).
Both bivalent anti-ICOS mAb alone (XENP16435) and XENP16435 in
combination with XENP16432 increase AKT phosphorylation
demonstrating ICOS ligation and agonism by XENP16435. Surprisingly,
despite only monovalent engagement of ICOS, treatment with the
anti-ICOS.times.anti-PD-1 bispecific antibodies induces more AKT
phosphorylation than treatment with XENP16435 alone and XENP16435
in combination with XENP16432. The positive AKT phosphorylation
data demonstrate a clear signature of ICOS activity with the
bispecific antibodies despite monovalent engagement of ICOS.
[0744] b. 5H(b): Activated T Helper Cell-Associated Genes
Upregulated by Anti-ICOS.times.Anti-PD-1 Bottle-Openers
[0745] Guedan et al. (2014) describes gene expression profiles for
activated T helper cells (i.e. Th1, Th2, and Th17) and regulatory T
cells (Tregs) following activation of ICOS signaling domain-based
CAR-Ts. They found that genes related to activated T helper cells
such as IL-17A, IL22, IFN?, TNF, and IL-13 were upregulated, while
Treg-related genes such as TGF.beta.1, SMAD3 and FOXP3 were either
unchanged or downregulated.
[0746] To investigate if engagement of T cells with
anti-ICOS.times.anti-PD-1 bispecific antibodies of the invention
led to a similar signature, we used Nanostring technology. PBMCs
were stimulated with 100 ng/mL SEB for 2 days. Cells were washed 2
times and restimulated with 100 ng/mL SEB and treated with
indicated test articles for 24 hours. RNA was extracted from cells
and assayed by nCounter.RTM. PanCancer Immune Profiling Panel
(NanoString Technologies, Seattle, Wash.) which assays 770 target
genes covering immune response. FIG. 52 depicts mean fold induction
in expression of a number of Th-related and Treg-related genes over
bivalent anti-RSV antibody. FIGS. 53A-53F respectively depict fold
induction of IL-17A, IL-17F, IL-22, IL-10, IL-9 and IFN? gene
expression by the indicated test articles over induction by
bivalent anti-RSV mAb.
[0747] As depicted in FIGS. 52-53 and consistent with the
observation by Gueden et al., Th-related genes associated with ICOS
signaling such as IL-17A, IL-17F, IL-22, IL-9, and IFN? are
upregulated following treatment with bivalent anti-ICOS mAb and
combination of anti-ICOS and anti-PD-1 mAbs. Notably, expression of
Th-related genes associated with ICOS signaling are further
upregulated following treatment with an anti-ICOS.times.anti-PD-1
antibody, again indicating a clear ICOS costimulatory signature and
dramatic synergy of anti-ICOS and anti-PD-1 engagement by a
bispecific antibody.
[0748] 9. 5I: Alternative Format Anti-ICOS.times.Anti-PD-1
Bispecific Antibodies
[0749] Alternative format costim/checkpoint bispecific antibodies
were produced to investigate whether the effect of
anti-ICOS.times.anti-PD-1 bottle-openers was unique to the
bottle-opener format or broadly applicable to
anti-ICOS.times.anti-PD-1 bispecific antibodies.
[0750] a. 5I(a): Anti-PD-1.times.Anti-ICOS Bottle-Opener
[0751] Anti-PD-1.times.anti-ICOS bottle-openers with an anti-PD-1
Fab generated using DNA encoding anti-PD-1 mAbs (e.g. XENP16432 and
XENP29120) as described in Example 2A and anti-ICOS scFvs as
described in Example 4B. Bottle-openers were produced as generally
described in Example 5A. Sequences for exemplary
anti-PD-1.times.anti-ICOS bottle-openers are depicted in FIG.
56.
[0752] b. 5I(b): Central-scFv
[0753] Schematics for the central-scFv format are depicted as FIG.
55A-B. DNA encoding anti-ICOS Fab-Fc heavy chains was generated
using DNA encoding anti-ICOS Fabs described in Example 4A by
standard subcloning into the expression vector pTT5. DNA encoding
anti-ICOS-anti-PD-1 Fab-scFv-Fc heavy chains was generated using
DNA encoding anti-ICOS Fabs described in Example 4A and anti-PD-1
scFv described in Example 2A by a combination of gene synthesis and
standard subcloning into the expression vector pTT5. DNA was
transfected into HEK293E cells for expression. Sequences for
exemplary anti-ICOS.times.anti-PD-1 central-scFv antibodies are
depicted in FIG. 57A-57C.
[0754] c. 5I(c): Central-scFv2
[0755] A schematic for the central-scFv2 format is depicted as FIG.
55C. DNA encoding anti-ICOS-anti-PD-1 Fab-scFv-Fc heavy chains was
generated using DNA encoding anti-ICOS Fabs described in Example 4A
and anti-PD-1 scFv described in Example 2A by a combination of gene
synthesis and standard subcloning into the expression vector pTT5.
DNA was transfected into HEK293E cells for expression. Sequences
for exemplary anti-ICOS.times.anti-PD-1 central-scFv2 antibodies
are depicted in FIG. 58.
[0756] d. 5I(d): Bispecific mAb
[0757] A schematic for the bispecific mAb format is depicted as
FIG. 2K. DNA encoding the anti-ICOS heavy and light chains were
generated based on the DNA encoding the anti-ICOS Fabs described in
Example, and DNA encoding anti-PD-1 heavy and light chains were
generated based on the DNA encoding the anti-PD-1 mAbs described in
Example 2A. Heavy chain VH genes were inserted via Gibson assembly
into pTT5 encoding the human IgG1 constant region with
E233P/L234V/L235A/G236del/S267K substitutions. Light chain VL genes
were inserted into pTT5 encoding the human C? constant region.
[0758] DNA was transfected into HEK293E cells for expression as 2
separate antibodies which are separately expressed and purified by
Protein A affinity (GE Healthcare). These antibodies contain
heterodimerization-skewing substitutions in the CH3:CH3 interface.
2-Mercaptoethylamine-HCl (2-MEA) was used to induce controlled
reduction of interchain disulfide bonds in the two parental IgGs.
2-MEA was then removed allowing the reoxidation of interchain
disulfide bonds to occur enabling recombination of the HC-LC pairs
(driven by the heterodimerization mutations). Finally, the
trispecific antibodies were purified by cation exchange
chromatography. Such methods are common in the art (see, for
instance, Labrijn (2013) PNAS 110(13):5145-50 or Strop et al.
(2012) J Mol Biol 420(3):204-19). Other methods of design and
purification known in the art may be used facilitate bispecific mAb
production, for instance, common light chain antibodies (Merchant
(1998) Nat Biotechnol 16(7):677-81) or heterodimeric Fab domains
(Lewis et al. (2014) Nat Biotechnol 32(2):191-8). Sequence for an
exemplary anti-PD-1.times.anti-ICOS bispecific mAb is depicted in
FIG. 59.
[0759] e. 5I(e): DVD-IgG
[0760] A schematic for the DVD-IgG format is depicted as FIG. 55E.
DNA encoding anti-PD-1-anti-ICOS VH-VH-CH1-Fc heavy chains and
VL-VL-C? light chains was generated using DNA encoding anti-ICOS
Fabs described in Example 4A and anti-PD-1 scFv described in
Example 2A by a combination of gene synthesis and standard
subcloning into the expression vector pTT5. DNA was transfected
into HEK293E cells for expression. Sequences for exemplary
anti-ICOS.times.anti-PD-1 DVD-IgGs are depicted in FIG. 60.
[0761] f. 5I(f): Trident
[0762] A schematic for the Trident format is depicted as FIG. 55F.
DNA encoding anti-PD-1 VL-VH-Fc heavy chains and VL-VH light chains
was generated using DNA encoding anti-ICOS Fabs described in
Example 4A and anti-PD-1 scFv described in Example 2A by a
combination of gene synthesis and standard subcloning into the
expression vector pTT5. DNA was transfected into HEK293E cells for
expression. Sequences for exemplary anti-ICOS.times.anti-PD-1
Tridents are depicted in FIG. 61A-61B.
[0763] g. 5I(g): Alternative Format Anti-ICOS.times.Anti-PD-1
Bispecific Antibodies Enhance Cytokine Secretion
[0764] Human PBMCs were stimulated with 200 ng/mL SEB for 2 days.
Cells were washed twice then re-stimulated with 100 ng/mL SEB and
treated with the indicated concentrations of the indicated test
articles. 24 hours after treatment, supernatants were assayed for
IL-2. The data are depicted in FIG. 62 and show that each of the
alternative format bispecific antibodies enhances IL-2 secretion in
comparison to bivalent anti-RSV (XENP15074) control. Notably, the
majority of these alternative format antibodies enhance IL-2
secretion in comparison to a bivalent anti-PD-1 antibody.
F. Example 6: Costim/Checkpoint Bispecific Antibodies Targeting
Different Immune Checkpoint Antigens
[0765] 1. 6A: Anti-CTLA-4.times.Anti-ICOS
[0766] Anti-CTLA-4.times.anti-ICOS bottle-openers were generated
with an anti-ICOS Fab derived from XENP16435 and anti-CTLA-4 scFvs
derived from ABDs described in Example 2B. Sequence for an
exemplary anti-ICOS.times.anti-CTLA-4 bottle-opener is depicted in
FIG. 63. Bispecific mAbs were produced as generally described in
Example 5I(c).
[0767] 2. 6B: Anti-LAG-3.times.Anti-ICOS
[0768] Anti-LAG-3.times.anti-ICOS bispecific antibodies were
generated with anti-LAG-3 Fabs derived from ABDs described in
Example 2C and an anti-ICOS Fab derived from XENP16435. Sequences
for exemplary anti-LAG-3.times.anti-ICOS bispecific antibodies are
depicted in FIG. 64A-64B. Bispecific mAbs were produced as
generally described in Example 5I(c).
[0769] 3. 6C: Anti-TIM-3.times.Anti-ICOS
[0770] Anti-TIM-3.times.anti-ICOS bispecific antibodies were
generated with anti-TIM-3 Fabs derived from ABDs described in
Example 2D and an anti-ICOS Fab derived from XENP16435. Sequence
for an exemplary anti-TIM-3.times.anti-ICOS bispecific antibody is
depicted in FIG. 65. Bispecific mAbs were produced as generally
described in Example 5I(c).
[0771] 4. 6D: Anti-PD-L1.times.Anti-ICOS
[0772] Anti-PD-L1.times.anti-ICOS bottle-openers were generated
with anti-PD-L1 Fabs derived from ABDs described in Example 2E and
anti-ICOS scFvs as described in Example 4B. Sequences for exemplary
anti-PD-L1.times.anti-ICOS bispecific antibodies are depicted in
FIG. 66A-66C. The bottle-openers were produced as generally
described in Example 5A.
[0773] 5. 6E: Additional Costim.times.Checkpoint Blockade
Bispecific Antibodies Function to Enhance Cytokine Secretion
[0774] Human PBMCs were stimulated with 200 ng/mL SEB for 2 days.
Cells were washed twice then restimulated with 100 ng/mL SEB and
treated with the indicated concentrations of the indicated test
articles (as described above). 24 hours after treatment,
supernatants were assayed for IL-2. The data are depicted in FIG.
67 and show that each of the additional costim.times.checkpoint
blockade bispecific antibodies enhance IL-2 secretion in comparison
to bivalent anti-RSV (XENP15074) control. Notably, the majority of
these alternative checkpoint blockade antibodies (but not all, e.g.
TIM-3 blockade) enhance IL-2 secretion in comparison to a bivalent
anti-PD-1 antibody (XENP16432).
G. Example 7: Monovalent Ligation of ICOS is Superior to Bivalent
Crosslinking
[0775] In Example 5A(a), it was surprisingly found that
anti-ICOS.times.anti-PD-1 antibody worked to enhance cytokine
secretion despite monovalent binding of ICOS which is contrary to
the crosslinking of costimulatory receptors such as ICOS which was
thought to be necessary for stimulation of cytokine production. In
Example 5H(a), it was surprisingly found that
anti-ICOS.times.anti-PD-1 antibodies enhanced AKT phosphorylation
(a signature of ICOS agonism) over bivalent anti-ICOS mAbs. In this
section, we further examine this trend in which monovalent binding
of ICOS appears to be superior to bivalent binding of ICOS.
[0776] 1. 7A: Production of One-Arm Anti-ICOS Fab-Fc Antibodies
[0777] Amino acid sequences for illustrative one-arm anti-ICOS
Fab-Fc antibodies are listed in FIGS. 68A-68G. DNA encoding the
three chains needed for antibody expression were generated by gene
synthesis and were subcloned using standard molecular biology
techniques into the expression vector pTT5. DNA was transfected
into HEK293E cells for expression and resulting proteins were
purified using standard techniques.
[0778] The resultant one-arm anti-ICOS Fab-Fc antibodies were
characterized for affinity to human ICOS using Octet. Anti-mouse Fc
(AMC) biosensors were used to capture mouse IgG2a Fc fusion of ICOS
and dipped into multiple concentrations of the test articles. The
resulting equilibrium dissociation constants (KD), association
rates (ka), and dissociation rates (kd) are presented in FIG. 69.
Binding affinities and kinetic rate constants were obtained by
analyzing the processed data using a 1:1 binding model using
ForteBio Octet Data Analysis software (ForteBio).
[0779] 2. 7B: Monovalent One-Arm Anti-ICOS Fab-Fc Antibodies
Promote Greater AKT Activation in PBMCs than Bivalent Anti-ICOS
Antibody
[0780] PBMCs were stimulated with 100 ng/mL SEB for 2 days.
Following stimulation, CD3+ T cells were isolated by negative
selection using EasySep.TM. Human T Cell Enrichment Kit (STEMCELL
Technologies, Vancouver, Canada) and then treated with indicated
test articles in combination with plate bound anti-CD3 antibody
(OKT3; 500 ng/mL). Cells were lysed 30 minutes after treatment and
assayed for total AKT and phosphorylated AKT (Ser473) by a
multiplexed phosphoprotein assay on MULTI-SPOT 384-Well Spot plates
(Meso Scale Discovery, Rockville, Md.). The data are depicted in
FIG. 70 as percentage of AKT phosphorylated following
treatment.
[0781] Consistent with Example X, the anti-ICOS.times.anti-PD-1
bispecific antibodies promoted significantly greater AKT activation
than bivalent anti-ICOS antibody. Surprisingly, monovalent one-arm
anti-ICOS Fab-Fc antibodies enhanced also promoted significantly
greater AKT activation than bivalent anti-ICOS antibody to a level
comparable to the bispecific antibodies.
[0782] 3. 7C: Monovalent Agonism of ICOS Works with Multiple
Anti-ICOS ABDs
[0783] CD3+ T cells were isolated by negative selection using
EasySep.TM. Human T Cell Enrichment Kit (STEMCELL Technologies,
Vancouver, Canada) and then treated with indicated test articles in
combination with plate bound anti-CD3 antibody (OKT3; 500 ng/mL).
Cells were lysed 30 minutes after treatment and assayed for total
AKT and phosphorylated AKT (Ser473) by a multiplexed phosphoprotein
assay on MULTI-SPOT.TM. 384-Well Spot plates (Meso Scale Discovery,
Rockville, Md.). The data is depicted in FIG. 74 and show that
monovalent anti-ICOS Fab-Fc antibodies comprising various anti-ICOS
ABDs were able to induce AKT phosphorylation.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210095030A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210095030A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References