U.S. patent application number 15/943821 was filed with the patent office on 2018-11-29 for novel bispecific antigen binding molecules capable of specific binding to cd40 and to fap.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Peter BRUENKER, Alexander BUJOTZEK, Harald DUERR, Guy GEORGES, Christian KLEIN, Stephane LECLAIR, Moritz RAPP, Eva Carina SUM, Christine TRUMPFHELLER, Pablo UMANA.
Application Number | 20180340030 15/943821 |
Document ID | / |
Family ID | 61913152 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180340030 |
Kind Code |
A1 |
BRUENKER; Peter ; et
al. |
November 29, 2018 |
NOVEL BISPECIFIC ANTIGEN BINDING MOLECULES CAPABLE OF SPECIFIC
BINDING TO CD40 AND TO FAP
Abstract
The invention relates to novel bispecific antigen binding
molecules, comprising (a) at least one antigen binding domain
capable of specific binding to CD40, and (b) at least one antigen
binding domain capable of specific binding to a target cell
antigen, in particular Fibroblast Activation Protein (FAP), and to
methods of producing these molecules and to methods of using the
same.
Inventors: |
BRUENKER; Peter; (Schlieren,
CH) ; BUJOTZEK; Alexander; (Muenchen, DE) ;
DUERR; Harald; (Starnberg, DE) ; GEORGES; Guy;
(Habach, DE) ; KLEIN; Christian; (Schlieren,
CH) ; LECLAIR; Stephane; (Gauting, DE) ; RAPP;
Moritz; (Schlieren, CH) ; SUM; Eva Carina;
(Schlieren, CH) ; TRUMPFHELLER; Christine;
(Schlieren, CH) ; UMANA; Pablo; (Schlieren,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
61913152 |
Appl. No.: |
15/943821 |
Filed: |
April 3, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/33 20130101; C07K 2317/64 20130101; C07K 2317/565
20130101; A61K 39/39558 20130101; C07K 2317/52 20130101; C07K
2317/92 20130101; C07K 2317/31 20130101; C07K 2317/56 20130101;
C07K 2317/24 20130101; C07K 2317/35 20130101; C07K 2317/569
20130101; C07K 2317/55 20130101; C07K 2317/567 20130101; C07K
16/2878 20130101; C07K 2317/71 20130101; C07K 2317/75 20130101;
C07K 2317/94 20130101; A61K 2039/572 20130101; A61P 35/00 20180101;
A61K 2039/5154 20130101; C07K 16/40 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 4, 2017 |
EP |
17164725.8 |
Feb 27, 2018 |
EP |
18158751.0 |
Claims
1. A bispecific antigen binding molecule, comprising (a) at least
one antigen binding domain capable of specific binding to CD40, and
(b) at least one antigen binding domain capable of specific binding
to a target cell antigen.
2. The bispecific antigen binding molecule of claim 1, additionally
comprising (c) a Fc region composed of a first and a second subunit
capable of stable association.
3. The bispecific antigen binding molecule of claim 1 or claim 2,
wherein the antigen binding domain capable of specific binding to
CD40 binds to a polypeptide comprising, or consisting of, the amino
acid sequence of SEQ ID NO:1.
4. The bispecific antigen binding molecule of any one of claims 1
to 3, wherein the antigen binding domain capable of specific
binding to a target cell antigen is an antigen binding domain
capable of specific binding to Fibroblast Activation Protein
(FAP).
5. The bispecific antigen binding molecule of any one of claims 1
to 4, wherein the antigen binding domain capable of specific
binding to FAP comprises (a) a heavy chain variable region
(V.sub.HFAP) comprising (i) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:3, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:4, and (iii) CDR-H3 comprising the amino acid
sequence of SEQ ID NO:5, and a light chain variable region
(V.sub.LFAP) comprising (iv) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:6, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:7, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:8, or (b) a heavy chain variable region
(V.sub.HFAP) comprising (i) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:11, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:12, and (iii) CDR-H3 comprising the amino
acid sequence of SEQ ID NO:13, and a a light chain variable region
(V.sub.LFAP) comprising (iv) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:14, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:15, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:16.
6. The bispecific antigen binding molecule of any one of claims 1
to 5, wherein the antigen binding domain capable of specific
binding to FAP comprises (a) a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:9, and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:10, or (b) a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:17, and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:18.
7. The bispecific antigen binding molecule of any one of claims 1
to 6, wherein the antigen binding domain capable of specific
binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising (i) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:19, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:20, and (iii) CDR-H3 comprising the amino
acid sequence of SEQ ID NO:21, and a light chain variable region
(V.sub.LCD40) comprising (iv) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:22, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:23, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:24.
8. The bispecific antigen binding molecule of any one of claims 1
to 6, wherein the antigen binding domain capable of specific
binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising (i) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:27, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:28, and (iii) CDR-H3 comprising the amino
acid sequence of SEQ ID NO:29, and a light chain variable region
(V.sub.LCD40) comprising (iv) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:30, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:31, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:32.
9. The bispecific antigen binding molecule of any one of claims 1
to 8, wherein the antigen binding domain capable of specific
binding to CD40 comprises (a) a VH comprising the amino acid
sequence of SEQ ID NO:25 and a VL comprising the amino acid
sequence of SEQ ID NO:26, or (b) a VH comprising the amino acid
sequence of SEQ ID NO:33 and a VL comprising the amino acid
sequence of SEQ ID NO:34.
10. The bispecific antigen binding molecule of any one of claims 1
to 7, wherein the antigen binding domain capable of specific
binding to CD40 comprises (i) a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173 and
SEQ ID NO:174, and (ii) a light chain variable region (V.sub.LCD40)
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ
ID NO:178.
11. The bispecific antigen binding molecule of any one of claims 1
to 7, wherein the antigen binding domain capable of specific
binding to CD40 comprises (i) a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181,
SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184, and (ii) a light
chain variable region (V.sub.LCD40) comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:185, SEQ
ID NO:186, SEQ ID NO:187, and SEQ ID NO:188.
12. The bispecific antigen binding molecule of any one of claim 1
to 7 or 10, wherein the antigen binding domain capable of specific
binding to CD40 comprises (a) a VH comprising the amino acid
sequence of SEQ ID NO:171 and a VL comprising the amino acid
sequence of SEQ ID NO:175, or (b) a VH comprising the amino acid
sequence of SEQ ID NO:173 and a VL comprising the amino acid
sequence of SEQ ID NO:177, or (c) a VH comprising the amino acid
sequence of SEQ ID NO:174 and a VL comprising the amino acid
sequence of SEQ ID NO:178, or (d) a VH comprising the amino acid
sequence of SEQ ID NO:171 and a VL comprising the amino acid
sequence of SEQ ID NO:177, or (e) a VH comprising the amino acid
sequence of SEQ ID NO:171 and a VL comprising the amino acid
sequence of SEQ ID NO:178, or (f) a VH comprising the amino acid
sequence of SEQ ID NO:173 and a VL comprising the amino acid
sequence of SEQ ID NO:175, or (g) a VH comprising the amino acid
sequence of SEQ ID NO:173 and a VL comprising the amino acid
sequence of SEQ ID NO:178, or (h) a VH comprising the amino acid
sequence of SEQ ID NO:174 and a VL comprising the amino acid
sequence of SEQ ID NO:175, or (i) a VH comprising the amino acid
sequence of SEQ ID NO:174 and a VL comprising the amino acid
sequence of SEQ ID NO:177, or (j) a VH comprising the amino acid
sequence of SEQ ID NO:171 and a VL comprising the amino acid
sequence of SEQ ID NO:176, or (k) a VH comprising the amino acid
sequence of SEQ ID NO:172 and a VL comprising the amino acid
sequence of SEQ ID NO:175, or (l) a VH comprising the amino acid
sequence of SEQ ID NO:172 and a VL comprising the amino acid
sequence of SEQ ID NO:176, or (m) a VH comprising the amino acid
sequence of SEQ ID NO:172 and a VL comprising the amino acid
sequence of SEQ ID NO:177, or (n) a VH comprising the amino acid
sequence of SEQ ID NO:172 and a VL comprising the amino acid
sequence of SEQ ID NO:178, or (o) a VH comprising the amino acid
sequence of SEQ ID NO:173 and a VL comprising the amino acid
sequence of SEQ ID NO:176, or (p) a VH comprising the amino acid
sequence of SEQ ID NO:174 and a VL comprising the amino acid
sequence of SEQ ID NO:176.
13. The bispecific antigen binding molecule of any one of claim 1
to 7 or 10 or 12, wherein the antigen binding domain capable of
specific binding to CD40 comprises a VH comprising the amino acid
sequence of SEQ ID NO:171 and a VL comprising the amino acid
sequence of SEQ ID NO:175.
14. The bispecific antigen binding molecule of any one of claim 1
to 7 or 11, wherein the antigen binding domain capable of specific
binding to CD40 comprises (a) a VH comprising the amino acid
sequence of SEQ ID NO:179 and a VL comprising the amino acid
sequence of SEQ ID NO:185, or (b) a VH comprising the amino acid
sequence of SEQ ID NO:180 and a VL comprising the amino acid
sequence of SEQ ID NO:185, or (c) a VH comprising the amino acid
sequence of SEQ ID NO:181 and a VL comprising the amino acid
sequence of SEQ ID NO:185, or (d) a VH comprising the amino acid
sequence of SEQ ID NO:182 and a VL comprising the amino acid
sequence of SEQ ID NO:185, or (e) a VH comprising the amino acid
sequence of SEQ ID NO:179 and a VL comprising the amino acid
sequence of SEQ ID NO:186, or (f) a VH comprising the amino acid
sequence of SEQ ID NO:180 and a VL comprising the amino acid
sequence of SEQ ID NO:186, or (g) a VH comprising the amino acid
sequence of SEQ ID NO:181 and a VL comprising the amino acid
sequence of SEQ ID NO:186, or (h) a VH comprising the amino acid
sequence of SEQ ID NO:182 and a VL comprising the amino acid
sequence of SEQ ID NO:186, or (i) a VH comprising the amino acid
sequence of SEQ ID NO:183 and a VL comprising the amino acid
sequence of SEQ ID NO:187, or (j) a VH comprising the amino acid
sequence of SEQ ID NO:183 and a VL comprising the amino acid
sequence of SEQ ID NO:188, or (k) a VH comprising the amino acid
sequence of SEQ ID NO:184 and a VL comprising the amino acid
sequence of SEQ ID NO:187, or (l) a VH comprising the amino acid
sequence of SEQ ID NO:184 and a VL comprising the amino acid
sequence of SEQ ID NO:188.
15. The bispecific antigen binding molecule of any one of claim 1
to 7 or 11 or 14, wherein the antigen binding domain capable of
specific binding to CD40 comprises a VH comprising the amino acid
sequence of SEQ ID NO:179 and a VL comprising the amino acid
sequence of SEQ ID NO:185 or wherein the antigen binding domain
capable of specific binding to CD40 comprises a VH comprising the
amino acid sequence of SEQ ID NO:182 and a VL comprising the amino
acid sequence of SEQ ID NO:185.
16. The bispecific antigen binding molecule of any one of claims 1
to 7, comprising (i) at least one antigen binding domain capable of
specific binding to CD40, comprising a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173,
SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID
NO:182, SEQ ID NO:183 and SEQ ID NO:184, and a light chain variable
region (V.sub.LCD40) comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID
NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187
and SEQ ID NO:188, and (ii) at least one antigen binding domain
capable of specific binding to FAP, comprising a heavy chain
variable region (V.sub.HFAP) comprising an amino acid sequence of
SEQ ID NO:9 and a light chain variable region (V.sub.LFAP)
comprising an amino acid sequence of SEQ ID NO:10, or a heavy chain
variable region (V.sub.HFAP) comprising an amino acid sequence of
SEQ ID NO:17 and a light chain variable region (V.sub.LFAP)
comprising an amino acid sequence of SEQ ID NO:18.
17. The bispecific antigen binding molecule of any one of claims 2
to 16, wherein the Fc region is an IgG, particularly an IgG1 Fc
region or an IgG4 Fc region and wherein the Fc region comprises one
or more amino acid substitution that reduces the binding affinity
of the antibody to an Fc receptor and/or effector function.
18. The bispecific antigen binding molecule of any one of claims 2
to 17, wherein the Fc region is (i) of human IgG1 subclass with the
amino acid mutations L234A, L235A and P329G (numbering according to
Kabat EU index), or (ii) of mouse IgG1 subclass with the amino acid
mutations D265A and P329G (numbering according to Kabat EU
index).
19. The bispecific antigen binding molecule of any one of claims 1
to 18, wherein the bispecific antigen binding molecule comprises
(a) at least two Fab fragments capable of specific binding to CD40
connected to a Fc region, and (b) one antigen binding domain
capable of specific binding to FAP connected to the C-terminus of
the Fc region.
20. The bispecific antigen binding molecule of any one of claims 1
to 19, wherein the bispecific antigen binding molecule comprises
(a) at least two Fab fragments capable of specific binding to CD40
connected to a Fc region, and (b) a cross-fab fragment capable of
specific binding to FAP connected to the C-terminus of the Fc
region.
21. The bispecific antigen binding molecule of any one of claims 1
to 19, wherein the bispecific antigen binding molecule comprises
four Fab fragments capable of specific binding to CD40.
22. Polynucleotide encoding the bispecific antigen binding molecule
of any one of claims 1 to 21.
23. An expression vector comprising the polynucleotide of claim
22.
24. A host cell comprising polynucleotide of claim 22 or the
expression vector of claim 23.
25. A method of producing a bispecific antigen binding molecule,
comprising culturing the host cell of claim 24 under conditions
suitable for the expression of the bispecific antigen binding
molecule, and isolating the bispecific antigen binding
molecule.
26. A pharmaceutical composition comprising the bispecific antigen
binding molecule of any one of claims 1 to 21 and at least one
pharmaceutically acceptable excipient.
27. The bispecific antigen binding molecule of any one of claims 1
to 21, or the pharmaceutical composition of claim 26, for use as a
medicament.
28. The bispecific antigen binding molecule of any one of claims 1
to 21, or the pharmaceutical composition of claim 26, for use (i)
in inducing immune stimulation by CD40 expressing
antigen-presenting cells (APCs), (ii) in stimulating tumor-specific
T cell response, (iii) in causing apoptosis of tumor cells, (iv) in
the treatment of cancer, (v) in delaying progression of cancer,
(vi) in prolonging the survival of a patient suffering from cancer,
(vii) in the treatment of infections.
29. The bispecific antigen binding molecule of any one of claims 1
to 21, or the pharmaceutical composition of claim 26, for use in
the treatment of cancer.
30. Use of the bispecific antigen binding molecule of any one of
claims 1 to 21, or the pharmaceutical composition of claim 26, in
the manufacture of a medicament for the treatment of cancer.
31. A method of treating an individual having cancer comprising
administering to the individual an effective amount of the
bispecific antigen binding molecule of any one of claims 1 to 21,
or the pharmaceutical composition of claim 26.
32. The bispecific antigen binding molecule according to any one of
claims 1 to 21 or the pharmaceutical composition according to claim
26 for use in the treatment of cancer, wherein the bispecific
antigen binding molecule is administered in combination with a
chemotherapeutic agent, radiation and/or other agents for use in
cancer immunotherapy.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from European Patent
Application No. 17164725.8, filed Apr. 4, 2017 and European Patent
Application No. 18158751.0, filed Feb. 27, 2018, the contents of
which are incorporated herein by reference in their entireties.
SEQUENCE LISTING
[0002] The present application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 8, 2018, is named P34214-US_SeqListing.txt and is 635,489
bytes in size.
FIELD OF THE INVENTION
[0003] The invention relates to novel bispecific antigen binding
molecules, comprising (a) at least one antigen binding domain
capable of specific binding to CD40, and (b) at least one antigen
binding domain capable of specific binding to a target cell
antigen. In particular, these bispecific antigen binding molecules
further comprise (c) a Fc region composed of a first and a second
subunit capable of stable association. The invention further
relates to methods of producing these molecules and to methods of
using the same.
BACKGROUND
[0004] Multiple molecular signals are required during the
generation of a potent adaptive immune response. Signal one
involves the binding of a T-cell antigen receptor (TCR) to its
cognate antigen presented on the surface of antigen-presenting
cells (APCs). Signal two consists of the engagement of
costimulatory receptors with their respective ligands between T
cells and APCs. One of the best studied and most important
costimulatory effectors is the tumor necrosis factor receptor
(TNFR) family member CD40 and its ligand CD40L (Elgueta R. et al.,
Immunol Rev. 2009; 229(1):152-72). Several members of the TNFR
family including CD40 function after initial T cell activation to
sustain APC and T cell responses and thus have pivotal roles in the
organization and function of the immune system (Watts T. H. (2005)
Annu. Rev. Immunol. 23, 23-68). The combination of different
costimulatory TNFR family members allows a sequential and transient
regulation of APC and T cell activation and survival resulting in
increased immune responses while maintaining tight control of APC
and T cell function. Depending on the disease condition,
stimulation via costimulatory TNF family members can exacerbate or
ameliorate diseases. Activation or blockade of TNFR family
costimulators shows promise for several therapeutic applications in
multiple fields including cancer, infectious disease,
transplantation, and autoimmunity.
[0005] Among several costimulatory molecules, the TNFR family
member CD40 plays a key role in triggering immune responses by
inducing maturation, survival, antigen presentation, cytokine
production, and expression of costimulatory molecules of APCs,
which then drive antigen-specific T cell responses and NK cell
activation by proinflammatory cytokines. CD40 regulates immune
responses against infections, tumors and self-antigens and its
expression has been demonstrated on the surface of APCs such as B
cells, dendritic cells (DCs), monocytes, and macrophages as well as
platelets, and cells of non-hematopoietic origin such as
myofibroblasts, fibroblasts, epithelial, and endothelial cells
(Elgueta R. et al., Immunol Rev. 2009; 229(1):152-72). The CD40
ligand CD40L is expressed on activated CD4.sup.+ helper T cells,
platelets, monocytic cells, natural killer cell, mast cells, and
basophils (Carbone E. et. al., J Exp Med. 1997; 185(12): 2053-2060,
or Elgueta R. et al., Immunol Rev. 2009; 229(1):152-72). Expression
of CD40 and CD40L is strongly upregulated in response to various
immune stimulatory signals and CD40-CD40L interaction between APCs
and CD4.sup.+ T cells contributes to increased APC activation and
antigen-specific CD8.sup.+ T cell responses (Bevan M J., Nat Rev
Immunol. 2014; 4(8):595-602). Similar immune stimulatory results
were observed by using CD40 agonistic antibodies (Vonderheide R H
and Glennie M J., Clin Cancer Res. 2013; 19(5):1035-43).
[0006] Engagement of the type I transmembrane receptor CD40 by its
natural ligand CD40L, a type II transmembrane protein or by
agonistic antibodies promotes CD40 clustering and induces the
recruitment of adapter proteins to the cytoplasmic receptor domain.
The recruitment of these adapter proteins known as TNF
receptor-associated factors (TRAFs) leads to synergistic activation
of mitogen-activated protein kinases (MAPKs), phosphoinositide
3-kinase (PI3K) as well as canonical and non-canonical nuclear
factor .kappa.B (NFKB) signaling pathways (Elgueta R. et al.,
Immunol Rev. 2009; 229(1):152-72). In turn, this results in APC
maturation and activation, which then maximizes antigen-specific T
cell responses. Recent studies have shown two different modes of
action of agonistic CD40 antibodies in harnessing anti-tumor
immunity. Beside its indirect mode of action by mediated tumor cell
killing through the activation of the adaptive immune system,
agonistic CD40 antibodies can induce direct tumor cell killing
through inducing apoptosis of CD40-expressing solid tumor cells
(Eliopoulos A G. et al., Mol Cell Biol. 2000; 20(15):5503-15). The
direct CD40 antibody-mediated killing of tumor cells can provide a
source of tumor antigens that can be processed and presented by APC
simultaneously activated by CD40 engagement via anti-CD40
antibodies which then can induce tumor antigen-specific T cells, a
postulated mechanism known as endogenous vaccination. Given that
CD40 engagement can mount in an efficient anti-cancer immune
response, agonistic CD40 antibodies have been used successfully in
a variety of preclinical tumor models, both as a single-agent and
in combination with chemotherapy (Vonderheide R H and Glennie M J.,
Clin Cancer Res. 2013; 19(5):1035-43).
[0007] To date, six CD40 mAb are under investigation in clinical
trials: Chi Lob 7/4 (CD40 agonistic IgG1 chimeric mAb; Cancer
Research UK; Chowdhury F. et al., Cancer Immunol Res. 2013;
2:229-40), ADC1013 (fully human, CD40 agonistic IgG1 antibody;
Alligator Bioscience and Johnson & Johnson; Mangsbo S M. et
al., Clin Cancer Res. 2015 Mar. 1; 21(5):1115-26), APX-005 (fully
humanized, CD40 agonistic IgG1 mAb; Apexigen; Bjorck P. et al. J
Immunother Cancer. 2015; 3(Suppl 2): P198), SEA-CD40 (CD40
agonistic IgG1 chimeric mAb; Seattle Genetics; Gardai S J. et al.
AACR 106th Annual Meeting 2015; April 18-22, abstract 2472), as
well as RO7009789 (fully human, CD40 super agonistic IgG2 mAb) are
investigated in clinical phase I studies, and dacetuzumab (CD40
partial agonistic IgG1 chimeric mAb; Seattle Genetics; Khubchandani
S. et al., Curr Opin Investig Drugs. 2009; 10:579-87) is
investigated in a clinical phase II study. Eligible patients for
these studies have solid tumors, classical Hodgkin lymphoma (HL),
diffuse large B-cell lymphoma (DLBCL), or indolent lymphoma
(including follicular lymphoma). Diverse activities ranging from
Fc-dependent cytotoxicity of CD40.sup.+ tumor cells via complement
mediated cytotoxicity (CMC) or antibody dependent cellular
cytoxicity (ADCC) to APC activation to induce anti-tumor T cell
responses as well as macrophage activation to deplete tumor and
tumor stroma have been shown for these CD40 agonistic antibodies.
So far there is no conclusive explanation for this observed
heterogeneity. However, recent studies indicate that this mode of
action diversity can be explained, at least in part, by differences
of the anti-CD40 antibodies in epitope specificity, isotype or
Fc:Fc.gamma.R interaction. For example, it appears that CD40
agonistic antibodies in vivo require crosslinking CD40, bound by
its Fab fragment on the target cell, to a Fc.gamma. receptor, bound
by its Fc fragment on a cell other than the target cell as has been
described for agonistic antibodies specific to other
apoptosis-inducing or immunomodulatory members of the
TNFR-superfamily (Dahan R., Cancer Cell. 2016 Jun. 13;
29(6):820-31; Li F. and Ravetch J. V. Science, 2011; 333,
1030-1034; Teng M. W. et al., J. Immunol. 2009; 183, 1911-1920).
The proposed mechanism includes Fc.gamma. receptor mediated
clustering of CD40 transmembrane molecules on target cells and
subsequent heightened CD40 signaling to achieve potent in vivo
efficacy.
[0008] The clinical development of agonistic CD40 antibodies has
provided promising initial results. In a first clinical trial
CP-870,893 has shown clinical efficacy in patients with advanced
cancer. Four out of 29 patients with advanced cancer showed partial
responses after receiving a single intravenous infusion of
CP-870,893 (Vonderheide R H., J Clin Oncol. 2007 Mar. 1;
25(7):876-83). One out of these four patients treated with 9
subsequent doses of CP-870,893 over one and a half years remained
in complete remission for more than 5 years. However, the most
common side effects of CP-870,893 are cytokine release syndromes
and thromboembolic events, so that with the dose schedules and
routes of administration used the combined data of the phase 1
clinical studies with more than 140 patients only indicates a
limited clinical efficacy and a local administration of the
antibody was suggested (Vonderheide R H, Glennie M, Clin Cancer
Res. 2013, 19(5), 1035-1043). The lack of single agent responses
occur in part due to severe on target/off tumor effects caused by
broad CD40 expression, which results in dose limiting toxicity
(e.g. cytokine release syndrome). The development of an agonistic
CD40 antibody that specifically activates APCs when CD40 is
cross-linked by a tumor-specific target could reduce side effects
and decrease dose limitations, offering new therapeutic options
with the potential to generate an efficient long lasting
anti-cancer immunity.
[0009] The available pre-clinical and clinical data clearly
demonstrate that there is a high clinical need for effective
agonists of CD40 that are able to induce and enhance effective
endogenous immune responses to cancer. However, almost never are
the effects limited to a single type of cells or acting via a
single mechanism and studies designed to elucidate inter- and
intracellular signaling mechanisms have revealed increasing levels
of complexity. Known CD40 antibodies can only be administered in
relatively low doses due to dose-limiting toxicities such as
cytokine release syndrome and thrombocyte/endothelial cell
activation, resulting in an insufficient activation of the pathway
on target APCs and a narrow therapeutic index. Thus, there is a
need of "targeted" agonists that preferably act on a single type of
cells.
[0010] The invention relates to new bispecific antigen binding
molecules capable of specific binding to CD40 and a target cell
antigen. The antigen binding molecules of the invention combine a
moiety capable of preferred binding to tumor-specific or
tumor-associated targets with a moiety capable of agonistic binding
to CD40, wherein the activation of APCs through CD40 is provided by
cross-linking through the target cell antigen, for example FAP
expressed on tumor stroma cells and potentially also through FAP
intermediately expressed in secondary lymphoid tissues. The
FAP-dependent cross-linking of the bispecific antigen binding
molecules confines the activation of CD40-expressing cells to the
tumor tissue and potentially also to secondary lymphoid tissues
such as tumor-draining lymph nodes. In contrast to bispecific
antigen binding molecules capable of specific binding to CD40 and
to immune checkpoint receptors on activated T cells, such as CTLA-4
or PD-1, targeting to a tumor target such as FAP enables
CD40-mediated APC activation mainly in the tumor stroma and
tumor-draining lymph nodes where fibroblasts express increased
levels of FAP compared to other tissues. The antigen binding
molecules of this invention may thus be able to trigger the CD40
receptor not only effectively, but also very selectively at the
desired site while overcoming the need for Fc.gamma.R cross-linking
thereby reducing side effects.
SUMMARY OF THE INVENTION
[0011] The present invention relates to bispecific antigen binding
molecules combining at least one moiety (antigen binding domain)
capable of specific binding to the costimulatory TNF receptor
family member CD40, with at least one antigen binding side
targeting a target cell antigen. These bispecific antigen binding
molecules are advantageous as they will preferably activate
costimulatory CD40 receptors at the site where the target cell
antigen is expressed, due to their binding capability towards a
target cell antigen.
[0012] In one aspect, the invention provides a bispecific antigen
binding molecule, comprising [0013] (a) at least one antigen
binding domain capable of specific binding to CD40, and [0014] (b)
at least one antigen binding domain capable of specific binding to
a target cell antigen.
[0015] In a particular aspect, the bispecific antigen binding
molecule comprises (a) at least one antigen binding domain capable
of specific binding to CD40, (b) at least one antigen binding
domain capable of specific binding to a target cell antigen, and
(c) a Fc domain composed of a first and a second subunit capable of
stable association. More particularly, the Fc domain composed of a
first and a second subunit capable of stable association comprises
mutations that reduce effector function.
[0016] In one aspect, the antigen binding domain capable of
specific binding to CD40 binds to a polypeptide comprising, or
consisting of, the amino acid sequence of SEQ ID NO:1.
[0017] In a further aspect, provided is a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to a target cell antigen is an antigen binding
domain capable of specific binding to Fibroblast Activation Protein
(FAP). In particular, the antigen binding domain capable of
specific binding to FAP binds to a polypeptide comprising, or
consisting of, the amino acid sequence of SEQ ID NO:2. Thus, in one
aspect, the invention provides a bispecific antigen binding
molecule, comprising (a) at least one antigen binding domain
capable of specific binding to CD40, and (b) at least one antigen
binding domain capable of specific binding to FAP.
[0018] In one aspect, the invention provides a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to FAP comprises
[0019] (a) a heavy chain variable region (V.sub.HFAP) comprising
(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (ii)
CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (iii)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:5, and a
light chain variable region (V.sub.LFAP) comprising (iv) CDR-L1
comprising the amino acid sequence of SEQ ID NO:6, (v) CDR-L2
comprising the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3
comprising the amino acid sequence of SEQ ID NO:8, or [0020] (b) a
heavy chain variable region (V.sub.HFAP) comprising (i) CDR-H1
comprising the amino acid sequence of SEQ ID NO:11, (ii) CDR-H2
comprising the amino acid sequence of SEQ ID NO:12, and (iii)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:13, and a a
light chain variable region (V.sub.LFAP) comprising (iv) CDR-L1
comprising the amino acid sequence of SEQ ID NO:14, (v) CDR-L2
comprising the amino acid sequence of SEQ ID NO:15, and (vi)
[0021] CDR-L3 comprising the amino acid sequence of SEQ ID
NO:16.
[0022] In a further aspect, provided is a bispecific antigen
binding molecule as defined herein before, wherein the antigen
binding domain capable of specific binding to FAP comprises [0023]
(a) a heavy chain variable region (V.sub.HFAP) comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO:9, and a
light chain variable region (V.sub.LFAP) comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO:10, or [0024] (b)
a heavy chain variable region (V.sub.HFAP) comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO:17, and a light
chain variable region (V.sub.LFAP) comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO:18.
[0025] In particular, provided is a bispecific antigen binding
molecule as defined herein before, wherein the antigen binding
domain capable of specific binding to FAP comprises (a) a heavy
chain variable region (V.sub.HFAP) comprising an amino acid
sequence of SEQ ID NO:9, and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence of SEQ ID NO:10, or
(b) a heavy chain variable region (V.sub.HFAP) comprising an amino
acid sequence of SEQ ID NO:17, and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence of SEQ ID NO:18.
[0026] In a further aspect, provided is a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising (i) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:19, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:20, and (iii) CDR-H3 comprising the amino
acid sequence of SEQ ID NO:21, and a light chain variable region
(V.sub.LCD40) comprising (iv) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:22, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:23, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:24.
[0027] In another aspect, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 binds to mouse CD40 and comprises a heavy chain
variable region (V.sub.HCD40) comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:27, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:28, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:29, and a light chain variable
region (V.sub.LCD40) comprising (iv) CDR-L1 comprising the amino
acid sequence of SEQ ID NO:30, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:31, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:32.
[0028] Furthermore, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 comprises (a) a VH comprising the amino acid
sequence of SEQ ID NO:25 and a VL comprising the amino acid
sequence of SEQ ID NO:26, or (b) a VH comprising the amino acid
sequence of SEQ ID NO:33 and a VL comprising the amino acid
sequence of SEQ ID NO:34. In a particular aspect, the invention
provides a bispecific antigen binding molecule, wherein each of the
antigen binding domains capable of specific binding to CD40
comprises a VH comprising the amino acid sequence of SEQ ID NO:25
and a VL comprising the amino acid sequence of SEQ ID NO:26.
[0029] In a further aspect, provided is a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising
[0030] (i) CDR-H1 comprising the amino acid sequence selected from
the group consisting of SEQ ID NO:19 and SEQ ID NO:35,
[0031] (ii) CDR-H2 comprising the amino acid sequence selected from
the group consisting of SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:37,
SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID
NO:42, SEQ ID NO:43 and SEQ ID NO:44, and
[0032] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:21, and a light chain variable region (V.sub.LCD40)
comprising
[0033] (iv) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:22,
[0034] (v) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:23, and
[0035] (vi) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:24.
[0036] In yet another aspect, provided is a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising
[0037] (i) CDR-H1 comprising the amino acid sequence selected from
the group consisting of SEQ ID NO:19 and SEQ ID NO:261,
[0038] (ii) CDR-H2 comprising the amino acid sequence selected from
the group consisting of
[0039] SEQ ID NO:20, SEQ ID NO:262 and SEQ ID NO:263, and
[0040] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:21, and a light chain variable region (V.sub.LCD40)
comprising
[0041] (iv) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:22, SEQ ID NO:264 and SEQ ID NO:265,
[0042] (v) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:23, and
[0043] (vi) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:24.
[0044] Furthermore, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 comprises
[0045] (i) a heavy chain variable region (V.sub.HCD40) comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:171, SEQ ID NO:172, SEQ ID NO:173 and SEQ ID NO:174, and
[0046] (ii) a light chain variable region (V.sub.LCD40) comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178.
[0047] In particular, a bispecific antigen binding molecule is
provided, wherein the antigen binding domain capable of specific
binding to CD40 comprises
[0048] (a) a VH comprising the amino acid sequence of SEQ ID NO:171
and a VL comprising the amino acid sequence of SEQ ID NO:175,
or
[0049] (b) a VH comprising the amino acid sequence of SEQ ID NO:173
and a VL comprising the amino acid sequence of SEQ ID NO:177,
or
[0050] (c) a VH comprising the amino acid sequence of SEQ ID NO:174
and a VL comprising the amino acid sequence of SEQ ID NO:178,
or
[0051] (d) a VH comprising the amino acid sequence of SEQ ID NO:171
and a VL comprising the amino acid sequence of SEQ ID NO:177,
or
[0052] (e) a VH comprising the amino acid sequence of SEQ ID NO:171
and a VL comprising the amino acid sequence of SEQ ID NO:178,
or
[0053] (f) a VH comprising the amino acid sequence of SEQ ID NO:173
and a VL comprising the amino acid sequence of SEQ ID NO:175,
or
[0054] (g) a VH comprising the amino acid sequence of SEQ ID NO:173
and a VL comprising the amino acid sequence of SEQ ID NO:178,
or
[0055] (h) a VH comprising the amino acid sequence of SEQ ID NO:174
and a VL comprising the amino acid sequence of SEQ ID NO:175,
or
[0056] (i) a VH comprising the amino acid sequence of SEQ ID NO:174
and a VL comprising the amino acid sequence of SEQ ID NO:177,
or
[0057] (j) a VH comprising the amino acid sequence of SEQ ID NO:171
and a VL comprising the amino acid sequence of SEQ ID NO:176,
or
[0058] (k) a VH comprising the amino acid sequence of SEQ ID NO:172
and a VL comprising the amino acid sequence of SEQ ID NO:175,
or
[0059] (l) a VH comprising the amino acid sequence of SEQ ID NO:172
and a VL comprising the amino acid sequence of SEQ ID NO:176,
or
[0060] (m) a VH comprising the amino acid sequence of SEQ ID NO:172
and a VL comprising the amino acid sequence of SEQ ID NO:177,
or
[0061] (n) a VH comprising the amino acid sequence of SEQ ID NO:172
and a VL comprising the amino acid sequence of SEQ ID NO:178,
or
[0062] (o) a VH comprising the amino acid sequence of SEQ ID NO:173
and a VL comprising the amino acid sequence of SEQ ID NO:176,
or
[0063] (p) a VH comprising the amino acid sequence of SEQ ID NO:174
and a VL comprising the amino acid sequence of SEQ ID NO:176.
[0064] More particularly, provided is a bispecific antigen binding,
wherein the antigen binding domain capable of specific binding to
CD40 comprises a VH comprising the amino acid sequence of SEQ ID
NO:171 and a VL comprising the amino acid sequence of SEQ ID
NO:175.
[0065] In a further aspect, provided is a bispecific antigen
binding molecule of any one of claims 1 to 7, wherein the antigen
binding domain capable of specific binding to CD40 comprises
[0066] (i) a heavy chain variable region (V.sub.HCD40) comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183
and SEQ ID NO:184, and
[0067] (ii) a light chain variable region (V.sub.LCD40) comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:185, SEQ ID NO:186, SEQ ID NO:187, and SEQ ID NO:188.
[0068] In particular, a bispecific antigen binding molecule is
provided, wherein the antigen binding domain capable of specific
binding to CD40 comprises
[0069] (a) a VH comprising the amino acid sequence of SEQ ID NO:179
and a VL comprising the amino acid sequence of SEQ ID NO:185,
or
[0070] (b) a VH comprising the amino acid sequence of SEQ ID NO:180
and a VL comprising the amino acid sequence of SEQ ID NO:185,
or
[0071] (c) a VH comprising the amino acid sequence of SEQ ID NO:181
and a VL comprising the amino acid sequence of SEQ ID NO:185,
or
[0072] (d) a VH comprising the amino acid sequence of SEQ ID NO:182
and a VL comprising the amino acid sequence of SEQ ID NO:185,
or
[0073] (e) a VH comprising the amino acid sequence of SEQ ID NO:179
and a VL comprising the amino acid sequence of SEQ ID NO:186,
or
[0074] (f) a VH comprising the amino acid sequence of SEQ ID NO:180
and a VL comprising the amino acid sequence of SEQ ID NO:186,
or
[0075] (g) a VH comprising the amino acid sequence of SEQ ID NO:181
and a VL comprising the amino acid sequence of SEQ ID NO:186,
or
[0076] (h) a VH comprising the amino acid sequence of SEQ ID NO:182
and a VL comprising the amino acid sequence of SEQ ID NO:186,
or
[0077] (i) a VH comprising the amino acid sequence of SEQ ID NO:183
and a VL comprising the amino acid sequence of SEQ ID NO:187,
or
[0078] (j) a VH comprising the amino acid sequence of SEQ ID NO:183
and a VL comprising the amino acid sequence of SEQ ID NO:188,
or
[0079] (k) a VH comprising the amino acid sequence of SEQ ID NO:184
and a VL comprising the amino acid sequence of SEQ ID NO:187,
or
[0080] (l) a VH comprising the amino acid sequence of SEQ ID NO:184
and a VL comprising the amino acid sequence of SEQ ID NO:188.
[0081] More particularly, provided is a bispecific antigen binding,
wherein the antigen binding domain capable of specific binding to
CD40 comprises a VH comprising the amino acid sequence of SEQ ID
NO:179 and a VL comprising the amino acid sequence of SEQ ID NO:185
or wherein the antigen binding domain capable of specific binding
to CD40 comprises a VH comprising the amino acid sequence of SEQ ID
NO:182 and a VL comprising the amino acid sequence of SEQ ID
NO:185.
[0082] In another aspect, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 comprises
[0083] (i) a heavy chain variable region (V.sub.HCD40) comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ
ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54
and SEQ ID NO:55, and
[0084] (ii) a light chain variable region (V.sub.LCD40) comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,
SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64.
[0085] Particularly, a bispecific antigen binding molecule is
provided, wherein each of the moieties capable of specific binding
to CD40 comprises a VH comprising the amino acid sequence of SEQ ID
NO:47 and a VL comprising the amino acid sequence of SEQ ID
NO:57.
[0086] Furthermore, provided is a bispecific antigen binding
molecule comprising
[0087] (i) at least one antigen binding domain capable of specific
binding to CD40, comprising a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173,
SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID
NO:182, SEQ ID NO:183 and SEQ ID NO:184, and a light chain variable
region (V.sub.LCD40) comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID
NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187
and SEQ ID NO:188, and
[0088] (ii) at least one antigen binding domain capable of specific
binding to FAP, comprising a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:9 and a
light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:10, or a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:17 and
a light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:18.
[0089] In another aspect, provided is a bispecific antigen binding
molecule comprising
[0090] (i) at least one antigen binding domain capable of specific
binding to CD40, comprising a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:25, SEQ ID NO:45, SEQ ID NO:46, SEQ
ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, and a
light chain variable region (V.sub.LCD40) comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:26, SEQ ID
NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ
ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, and
[0091] (ii) at least one antigen binding domain capable of specific
binding to FAP, comprising a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:9 and a
light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:10, or a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:17 and
a light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:18.
[0092] In one aspect, the bispecific antigen binding molecule is a
humanized or a chimeric antibody. In a further aspect, the
bispecific antigen binding molecule comprises an IgG Fc region,
particularly an IgG1 Fc region or an IgG4 Fc region. In particular,
the Fc region comprises one or more amino acid substitution that
reduces the binding affinity of the antibody to an Fc receptor
and/or effector function. In a particular aspect, provided is a
bispecific antigen binding molecule, wherein the Fc region is (i)
of human IgG1 subclass with the amino acid mutations L234A, L235A
and P329G (numbering according to Kabat EU index), or (ii) of mouse
IgG1 subclass with the amino acid mutations D265A and P329G
(numbering according to Kabat EU index). Particularly, the Fc
region is of human IgG1 subclass with the amino acid mutations
L234A, L235A and P329G (numbering according to Kabat EU index).
[0093] In another aspect, provided is a bispecific antigen binding
molecule as defined herein before, wherein the first subunit of the
Fc region comprises knobs and the second subunit of the Fc region
comprises holes according to the knobs into holes method. In
particular, provided is a bispecific antigen binding molecule,
wherein (i) the first subunit of the Fc region comprises the amino
acid substitutions S354C and T366W (numbering according to Kabat EU
index) and the second subunit of the Fc region comprises the amino
acid substitutions Y349C, T366S and Y407V (numbering according to
Kabat EU index), or (ii) the first subunit of the Fc region
comprises the amino acid substitutions K392D and K409D (numbering
according to Kabat EU index) and the second subunit of the Fc
region comprises the amino acid substitutions E356K and D399K
(numbering according to Kabat EU index). More particularly,
provided is a bispecific antigen binding molecule, wherein the
first subunit of the Fc region comprises the amino acid
substitutions S354C and T366W (numbering according to Kabat EU
index) and the second subunit of the Fc region comprises the amino
acid substitutions Y349C, T366S and Y407V (numbering according to
Kabat EU index).
[0094] In a further aspect, provided is a bispecific antigen
binding molecule, wherein the bispecific antigen binding molecule
comprises
[0095] (a) at least two Fab fragments capable of specific binding
to CD40 connected to a Fc region, and
[0096] (b) at least one antigen binding domain capable of specific
binding to FAP connected to the C-terminus of the Fc region.
[0097] In another aspect, provided is a bispecific antigen binding
molecule, wherein the bispecific antigen binding molecule
comprises
[0098] (a) at least two Fab fragments capable of specific binding
to CD40 connected to a Fc region, and
[0099] (b) one antigen binding domain capable of specific binding
to FAP connected to the C-terminus of the Fc region.
[0100] In a particular aspect, the antigen binding domain capable
of specific binding to FAP connected to the C-terminus of the Fc
region is a cross-fab fragment. Thus, provided is a bispecific
antigen binding molecule, wherein the bispecific antigen binding
molecule comprises
[0101] (a) at least two Fab fragments capable of specific binding
to CD40 connected to a Fc region, and
[0102] (b) a cross-fab fragment capable of specific binding to FAP
connected to the C-terminus of the Fc region.
[0103] In one aspect, provided is a bispecific antigen binding
molecule comprising
[0104] (a) two light chains and two heavy chains of an antibody
comprising two Fab fragments capable of specific binding to CD40,
and a Fc region, and
[0105] (b) a VH and a VL of an antigen binding domain capable
specific binding to FAP, wherein the VH is connected to the
C-terminus of one of the two heavy chains of (a), and wherein the
VL is connected to the C-terminus of the other of the two heavy
chains of (a).
[0106] In another aspect, provided is a bispecific antigen binding
molecule comprising
[0107] (a) two light chains and two heavy chains of an antibody
comprising two Fab fragments capable of specific binding to CD40,
and a Fc region, and
[0108] (b) a cross-fab fragment capable specific binding to FAP,
wherein the VH-CL chain is connected to the C-terminus of one of
the two heavy chains of (a).
[0109] In yet another aspect, provided is a bispecific antigen
binding molecule comprising
[0110] (a) two light chains and two heavy chains of an antibody
comprising two Fab fragments capable of specific binding to CD40,
and a Fc region, and
[0111] (b) a cross-fab fragment capable specific binding to FAP,
wherein the VL-CH1 chain is connected to the C-terminus of one of
the two heavy chains of (a).
[0112] Furthermore, provided is a bispecific antigen binding
molecule comprising
[0113] (a) two light chains and two heavy chains of an antibody
comprising two Fab fragments capable of specific binding to CD40,
and a Fc region, and
[0114] (b) two Fab fragments capable of specific binding to FAP,
wherein one of the Fab fragments is connected to the C-terminus of
one of the two heavy chains of (a), and the other of the Fab
fragments is connected to the C-terminus of the other of the two
heavy chains of (a).
[0115] In another aspect, the invention provides a bispecific
antigen binding molecule comprising
[0116] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40
and a Fc region subunit,
[0117] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0118] (c) a VH and a VL of an antigen binding domain capable of
specific binding to FAP, wherein the VH is connected to the
C-terminus of one of the two heavy chains of (a), and wherein the
VL is connected to the C-terminus of the other of the two heavy
chains of (a).
[0119] In a further aspect, provided is a bispecific antigen
binding molecule comprising
[0120] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40,
and a Fc region subunit,
[0121] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0122] (c) two Fab fragments capable of specific binding to FAP,
wherein one of the Fab fragments is connected to the C-terminus of
one of the two heavy chains of (a), and the other of the Fab
fragments is connected to the C-terminus of the other of the two
heavy chains of (a).
[0123] In another aspect, provided is a bispecific antigen binding
molecule, wherein the bispecific antigen binding molecule
comprises
[0124] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40,
and a Fc region subunit,
[0125] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0126] (c) one Fab fragment capable of specific binding to FAP,
wherein the Fab fragments is connected to the C-terminus of one of
the two heavy chains of (a).
[0127] In another aspect, the Fab fragment or the two Fab fragments
capable of specific binding to FAP are crossover Fab fragments each
comprising a VL-CH1 chain and a VH-CL chain, and wherein the VH-CL
chain or the VL-CH1 chain is connected to the C-terminus of one of
the two heavy chains of (a).
[0128] In one aspect, provided is a bispecific antigen binding
molecule, wherein the bispecific antigen binding molecule comprises
four Fab fragments capable of specific binding to CD40. In a
particular aspect, provided is a bispecific antigen binding
molecule, wherein each of the two heavy chains of (a) as defined
herein before comprises two VH-CH1 chains of a Fab fragment capable
of specific binding to CD40 that are connected to each other,
optionally by a peptide linker.
[0129] In another aspect, the invention provides a bispecific
antigen binding molecule comprising
[0130] (a) two heavy chains, each heavy chain comprising two VH-CH1
chains of a Fab fragment capable of specific binding to CD40 that
are connected to each other, optionally by a peptide linker, and a
Fc region subunit,
[0131] (b) four light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0132] (c) a VH and a VL of an antigen binding domain capable of
specific binding to FAP, wherein the VH is connected to the
C-terminus of one of the two heavy chains of (a), and wherein the
VL is connected to the C-terminus of the other of the two heavy
chains of (a).
[0133] In another aspect, provided is a bispecific antigen binding
molecule, wherein the bispecific antigen binding molecule
comprises
[0134] (a) two heavy chains, each heavy chain comprising two VH-CH1
chains of a Fab fragment capable of specific binding to CD40 that
are connected to each other, optionally by a peptide linker, and a
Fc region subunit,
[0135] (b) four light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0136] (c) one Fab fragment or cross-Fab fragment capable of
specific binding to FAP, wherein the Fab or cross-Fab fragment is
connected to the C-terminus of one of the two heavy chains of
(a).
[0137] In another aspect, provided is a bispecific antigen binding
molecule comprising
[0138] (a) two heavy chains, each heavy chain comprising two VH-CH1
chains of a Fab fragment capable of specific binding to CD40 that
are connected to each other, optionally by a peptide linker, and a
Fc region subunit,
[0139] (b) four light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0140] (c) a cross-fab fragment capable specific binding to FAP,
wherein the VH-CL chain of said cross-fab fragment is connected to
the C-terminus of one of the two heavy chains of (a).
[0141] In yet another aspect, provided is a bispecific antigen
binding molecule comprising
[0142] (a) two heavy chains, each heavy chain comprising two VH-CH1
chains of a Fab fragment capable of specific binding to CD40 that
are connected to each other, optionally by a peptide linker, and a
Fc region subunit,
[0143] (b) four light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0144] (c) a cross-fab fragment capable specific binding to FAP,
wherein the VL-CH1 chain of said cross-fab fragment is connected to
the C-terminus of one of the two heavy chains of (a).
[0145] In another particular aspect, provided is a bispecific
antigen binding molecule, wherein one or more of the Fab fragments
capable of specific binding to CD40 comprises a CL domain
comprising an arginine (R) at amino acid at position 123 (numbering
according to Kabat EU index) and a lysine (K) at amino acid at
position 124 (numbering according to Kabat EU index), and a CH1
domain comprising a glutamic acid (E) at amino acid at position 147
(numbering according to Kabat EU index) and a glutamic acid (E) at
amino acid at position 213 (numbering according to Kabat EU
index).
[0146] According to another aspect of the invention, there is
provided an isolated polynucleotide encoding a bispecific antigen
binding molecule as described herein before. The invention further
provides a vector, particularly an expression vector, comprising
the isolated polynucleotide of the invention and a host cell
comprising the isolated polynucleotide or the expression vector of
the invention. In some aspects the host cell is a eukaryotic cell,
particularly a mammalian cell.
[0147] In another aspect, provided is a method of producing a
bispecific antigen binding molecule as described herein before,
comprising culturing the host cell as described above under
conditions suitable for the expression of the bispecific antigen
binding molecule, and isolating the bispecific antigen binding
molecule. The invention also encompasses the bispecific antigen
binding molecule that specifically binds to CD40 and to FAP
produced by the method of the invention.
[0148] The invention further provides a pharmaceutical composition
comprising a bispecific antigen binding molecule as described
herein before and at least one pharmaceutically acceptable
excipient.
[0149] Also encompassed by the invention is the bispecific antigen
binding molecule as described herein before, or the pharmaceutical
composition comprising the bispecific antigen binding molecule, for
use as a medicament.
[0150] In one aspect, provided is a bispecific antigen binding
molecule as described herein before or the pharmaceutical
composition of the invention, for use [0151] (i) in inducing immune
stimulation by CD40 expressing antigen-presenting cells (APCs),
[0152] (ii) in stimulating tumor-specific T cell response, [0153]
(iii) in causing apoptosis of tumor cells, [0154] (iv) in the
treatment of cancer, [0155] (v) in delaying progression of cancer,
[0156] (vi) in prolonging the survival of a patient suffering from
cancer, [0157] (vii) in the treatment of infections.
[0158] In a specific aspect, provided is the bispecific antigen
binding molecule as described herein before or the pharmaceutical
composition of the invention, for use in the treatment of cancer.
In another specific aspect, the invention provides the bispecific
antigen binding molecule as described herein before for use in the
treatment of cancer, wherein the bispecific antigen binding
molecule is administered in combination with a chemotherapeutic
agent, radiation and/or other agents for use in cancer
immunotherapy. In another aspect, provided is the bispecific
antigen binding molecule as described herein before or the
pharmaceutical composition of the invention, for use in
up-regulating or prolonging cytotoxic T cell activity.
[0159] In a further aspect, the invention provides a method of
inhibiting the growth of tumor cells in an individual comprising
administering to the individual an effective amount of the
bispecific antigen binding molecule as described herein before, or
the pharmaceutical composition of the invention, to inhibit the
growth of the tumor cells. In another aspect, the invention
provides a method of treating or delaying cancer in an individual
comprising administering to the individual an effective amount of
the bispecific antigen binding molecule as described herein before,
or the pharmaceutical composition of the invention.
[0160] Also provided is the use of the bispecific antigen binding
molecule as described herein before for the manufacture of a
medicament for the treatment of a disease in an individual in need
thereof, in particular for the manufacture of a medicament for the
treatment of cancer, as well as a method of treating a disease in
an individual, comprising administering to said individual a
therapeutically effective amount of a composition comprising the
bispecific antigen binding molecule of the invention in a
pharmaceutically acceptable form. In a specific aspect, the disease
is cancer. In any of the above aspects the individual is a mammal,
particularly a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0161] FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, FIG. 1F show
schematic representations of the bispecific antigen binding
molecules which specifically bind to human CD40 and to FAP. FIG. 1A
shows a schematic representation of a bispecific CD40-FAP antibody
in the 4+1 format consisting of four CD40 binding Fab domains
combined with one FAP binding moiety with VH at the C-terminus of
one heavy chain and VL at the C-terminus of the other heavy chain
(tetravalent for CD40 and monovalent for FAP). The black point
symbolizes knob-into-hole mutations. FIG. 1B shows a schematic
representation of a bispecific CD40-FAP antibody in the 4+2 format
consisting of four CD40 binding Fab domains combined with two FAP
binding Fab domains fused each at the C-terminus of the heavy
chains (tetravalent for CD40 and bivalent for FAP). FIG. 1C shows a
schematic representation of a bispecific CD40-FAP antibody in the
2+1 format consisting of two CD40 binding Fab domains combined with
one FAP binding moiety with VH at the C-terminus of one heavy chain
and VL at the C-terminus of the other heavy chain (bivalent for
CD40 and monovalent for FAP). The black point symbolizes
knob-into-hole mutations. FIG. 1D shows a schematic representation
of a bispecific CD40-FAP antibody in the 2+2 format consisting of
two CD40 binding Fab domains combined with two FAP binding Fab
domains fused each at the C-terminus of the heavy chains (bivalent
for CD40 and bivalent for FAP). FIG. 1E shows a schematic
representation of a bispecific CD40-FAP antibody in the 2+1 format
consisting of two CD40 binding Fab domains combined with one FAP
binding Fab domains fused at the C-terminus of one of the heavy
chains (bivalent for CD40 and monovalent for FAP). The black point
symbolizes knob-into-hole mutations. FIG. 1F shows a schematic
representation of a bispecific CD40-FAP antibody in the 1+1 format
consisting of one CD40 binding arm combined with one FAP binding
arm (monovalent for CD40 and monovalent for FAP). The black point
symbolizes knob-into-hole mutations.
[0162] FIG. 2A and FIG. 2B show the binding of human tetravalent
anti-CD40 antibodies in a FAP-targeted monovalent or bivalent
format to FAP negative tumor cells (FIG. 2A) and FAP positive tumor
cells (FIG. 2B). The transgenic modified mouse embryonic fibroblast
NIH/3T3-huFAP clone 19 expresses high levels of human fibroblast
activation protein (huFAP), whereas the parental cell line
NIH/3T3-wt expresses no huFAP. Only the tetravalent anti-CD40
antigen binding molecules with either one or two FAP binding
moieties but not the non-FAP targeted formats efficiently bind to
NIH/3T3-huFAP cells (FIG. 2B). The bivalent FAP construct binds
stronger than the monovalent construct. In contrast, no binding of
the FAP-targeted anti-CD40 antibodies to the NIH/3T3-wt cells was
detected (FIG. 2A). Shown is the binding as median of fluorescence
intensity (MFI) of phycoerythrin (PE)-labeled anti-human IgG
Fc.gamma.-specific goat IgG F(ab')2 fragment which is used as
secondary detection antibody. MFI was measured by flow cytometry.
The x-axis shows the concentration of antibody constructs.
[0163] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG.
3G, FIG. 3H show the in vitro activation of human B cells by mono-
or bivalent FAP-targeted anti-CD40 constructs. With NIH/3T3-FAP
cells the bispecific antibody monovalent for FAP induced a similar
increase of B cell activation marker expression (CD70, CD80, CD83,
and CD86) as the bivalent FAP-targeted molecule. Moreover, the B
cell activation marker upregulation by FAP-targeted bispecific
antigen-binding molecules was comparable to the upregulation
induced by the FAP-independent positive control antibodies. In the
absence of FAP (NIH/3T3-wt cells) no increase of B cell activation
markers could be observed with the bispecific antigen binding
molecules, while positive control antibodies induced an
upregulation of activation marker. Shown is the percentage of CD70
(FIG. 3A and FIG. 3B), CD80 (FIG. 3C and FIG. 3D), CD83 (FIG. 3E
and FIG. 3F) and CD86 (FIG. 3G and FIG. 3H) positive vital B cells
after 2 days incubation with the indicated titrated antibodies. The
x-axis shows the concentration of antibody constructs. The effect
on NIH/3T3-FAP cells is shown In FIG. 3A, FIG. 3C, FIG. 3E and FIG.
3G, respectively, while the effect on NIH/3T3-wt cells is shown in
FIG. 3B, FIG. 3D, FIG. 3F and FIG. 3H, respectively.
[0164] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F and
FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG.
5H show the in vitro activation of human B cells by mono- or
bivalent FAP-targeted human anti-CD40 constructs in the presence of
FAP-coated or uncoated Dynabeads.RTM. after 2 days (FIG. 4A, FIG.
4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F) or 5 days incubation (FIG.
5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H).
After 2 days incubation with FAP-coated beads the bispecific
antibodies monovalent for FAP induced a similar increase of B cell
activation marker expression (CD70, CD83, and CD86) as the bivalent
FAP-targeted molecules. Moreover, the B cell activation marker
upregulation by FAP-targeted bispecific antigen-binding molecules
was comparable to the upregulation induced by the FAP-independent
positive control antibodies. In the absence of FAP (uncoated beads)
no increase of B cell activation markers could be observed with the
bispecific antigen binding molecules, while positive control
antibodies induced an upregulation of activation markers. After 5
days B cell incubation with FAP-coated Dynabeads.RTM. FAP-targeted
human anti-CD40 constructs induced a FAP-dependent upregulation of
CD80 and CD86 expression on B cells. Compared to the
FAP-independent upregulation of CD86 induced by RO7009789 or
cross-linked SGN-40, CD86 upregulation induced by FAP-dependent
bispecific antigen binding molecules was slightly lower. For CD70
and CD83 no or only very limited upregulation could be observed
with the bispecifc antibodies targeting FAP and CD40, while the
positive control antibodies clearly showed an effect on these B
cell activation markers. Shown is the percentage of CD70 (FIG. 4A,
FIG. 4B, FIG. 5A and FIG. 5B, respectively), CD80 (FIG. 5C and FIG.
5D, respectively), CD83 (FIG. 4C, FIG. 4D, FIG. 5E and FIG. 5F,
respectively) and CD86 (FIG. 4E, FIG. 4F, FIG. 5G and FIG. 5H,
respectively) positive vital B cells after 2 days or 5 days
incubation with the indicated titrated antibodies. The x-axis shows
the concentration of antibody constructs.
[0165] FIG. 6A and FIG. 6B show the IL-6 secretion of human B cells
treated with different FAP-targeted and untargeted agonistic
anti-CD40 antibodies in the presence of FAP-coated (FIG. 6A) or
uncoated beads (FIG. 6B) after 5 days incubation. In the presence
of FAP the monovalent as well as the bivalent FAP-targeted
anti-CD40 antibody induced a similar increased IL-6 secretion as
compared to the FAP-independent positive control antibodies
RO7009789 and SGN40. In contrast, B cells treated with the
untargeted negative control antibodies expressed similar low IL-6
levels as untreated B cells. In the absence of FAP (uncoated beads)
no increase in IL-6 production was detected with the bispecific
antigen binding molecules. Shown is the IL-6 amount in the
supernatant of human B cells cultured for five days with the
indicated titrated antibodies measured by ELISA. The x-axis shows
the concentration of antibody constructs.
[0166] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG.
7G, FIG. 7H show the in vitro activation of human monocyte-derived
DCs (moDCs) by mono- or bivalent FAP-targeted human anti-CD40
constructs in the presence of FAP-coated or uncoated Dynabeads.RTM.
after 2 days incubation. In the presence of FAP-coated beads the
bispecific antibody monovalent for FAP induced a similar increase
of DC activation marker expression (CD70, CD80, and CD83) as the
bivalent FAP-targeted molecule, whereas in the absence of FAP
(uncoated beads) no activation marker upregulation was detected.
Moreover, the upregulation of CD80 and CD83 by FAP-targeted
bispecific antigen-binding molecules was comparable to the
upregulation induced by the FAP-independent positive control
antibodies. A higher upregulation of CD70 on DCs was observed for
RO709789 compared to all other tested antibodies. In contrast, CD86
expression was not significantly changed on DCs incubated with the
different anti-CD40 antibodies compared to untreated DCs. Shown is
the percentage of CD70 (FIG. 7A and FIG. 7B), CD80 (FIG. 7C and
FIG. 7D), CD83 (FIG. 7E and FIG. 7F) and CD86 (FIG. 7G and FIG. 7H)
positive vital moDCs after 2 days incubation with the indicated
titrated antibodies. The x-axis shows the concentration of antibody
constructs.
[0167] FIG. 8A and FIG. 8B show the in vitro activation of
HEK-Blue.TM. CD40L cells by mono- or bivalent FAP-targeted human
anti-CD40 constructs in the presence of FAP-coated or uncoated
Dynabeads.RTM. after 8 hours incubation. In the presence of
FAP-coated beads the bispecific antibody monovalent for FAP and
bivalent for CD40 induced a similar increase of SEAP production as
the bispecific antibody bivalent for FAP and CD40, whereas in the
absence of FAP (uncoated beads) no SEAP production was detected.
Moreover, an upregulation of SEAP production by FAP-targeted
antibodies tetravalent for CD40 was observed in the presence of
FAP. However, SEAP production was also observed in the absence of
FAP in the supernatant of reporter cells treated with FAP-targeted
antibodies tetravalent for CD40. The negative control antibodies
tetravalent for human CD40 with one or two DP47 domains instead of
a FAP binding domain induced comparable SEAP production in
HEK-Blue.TM. CD40L cells in the presence and absence of FAP and the
positive control antibody SGN-40+F(ab) induced similar levels of
SEAP production as compared to FAP-targeted bispecific antibodies
bivalent or tetravalent for human CD40 in the presence of
FAP-coated beads. Shown is the absorption at a wavelength of 650 nm
which correlates with the amount of hydrolyzed substrate by SEAP.
The x-axis shows the concentration of antibody constructs.
[0168] FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG.
9G, FIG. 9H show the T cell priming of SIINFEKL-pulsed DCs
activated by FAP-targeted anti-CD40 binding molecules. DCs isolated
from huCD40 transgenic mice (similar expression pattern of human
CD40 and mouse CD40), pulsed with low amounts of SIINFEKL and
stimulated with FAP-dependent bispecific anti-CD40 antibodies as
well as FAP-coated beads induced a strong proliferation of
antigen-specific T cells. In contrast, in the absence of FAP
(uncoated beads) no T cell proliferation was induced by DCs
stimulated with FAP-targeted anti-CD40 antibodies. T cell
proliferation levels induced by DCs stimulated with the murine or
the human bispecific antigen binding molecules with four CD40 and
two FAP binding moieties was comparable. No significant
upregulation of the T cell activation markers CD44 and CD25 or
IFN.gamma. production was observed for T cells co-cultured with DCs
pre-stimulated with different agonistic anti-CD40 antibodies. Only
DCs pulsed with high amounts of SIINFEKL induced a clear expression
increase of these markers compared to the untreated condition.
Shown is the percentage of proliferating (CFSE-low), IFN.gamma.,
CD25, and CD44 positive vital CFSE-labeled murine
CD3.sup.+CD8.sup.+ OT-1 T cells co-cultured with huCD40 tg DCs
pre-incubated with the indicated titrated antibodies. The x-axis
shows the concentration of antibody constructs.
[0169] FIG. 9I and FIG. 9J show the concentration of IL-2 and
IL-12(p40) measured in the supernatant of T cell primed by
SIINFEKL-pulsed FAP-targeted anti-CD40 antibody-activated DCs. In
the co-culture of OT-1 T cells and huCD40 tg DCs pulsed with low
amounts of SIINFEKL and stimulated with FAP-dependent bispecific
anti-CD40 antibodies as well as FAP-coated beads increased IL-2 and
IL-12(p40) levels were detected compared to OT-1 T cells
co-cultured with huCD40 tg DCs pre-stimulated with FAP-targeted
antibodies in the absence of FAP. Moreover, the murine bivalent
FAP-targeted anti-CD40 antibody induced a markedly higher secretion
of IL-12(p40) as the human equivalent bispecific antigen binding
molecule. IL-2 secretion was increased to a similar extent with
both, the anti-human CD40 and the anti-mouse CD40 bispecific
antigen binding molecules in a FAP-dependent way. Shown is the IL-2
and IL-12(p40) amount in the supernatant of murine
CD3.sup.+CD8.sup.+ OT-1 T cells co-cultured with huCD40 tg DCs
pre-incubated with the indicated titrated antibodies measured by
ELISA. The x-axis shows the concentration of antibody
constructs.
[0170] FIG. 10A, FIG. 10B, FIG. 10C, FIG. 11A, FIG. 11B, FIG. 11C,
FIG. 12A, FIG. 12B, FIG. 12C show the T cell priming of OVA-pulsed
DCs activated by FAP-targeted anti-CD40 binding molecules. DCs
isolated from huCD40 transgenic mice, treated with DEC205-OVA
conjugate and stimulated with FAP-dependent bispecific anti-CD40
antibodies as well as FAP-coated beads induced a strong
proliferation and CD25 as well as CD44 expression of
antigen-specific T cells. In contrast, in the absence of FAP
(uncoated beads) or OVA (DEC only) no T cell proliferation and
activation was induced by DCs stimulated with FAP-targeted
anti-CD40 antibodies. T cell proliferation and CD25 as well as CD44
expression levels induced by DCs stimulated with the murine or the
human bispecific antigen binding molecules with four CD40 and two
FAP binding moieties was comparable. DCs pulsed with high amounts
of SIINFEKL instead of DEC205-OVA conjugate also induced a strong T
cell proliferation and expression of the activation markers CD25
and CD44. Shown is the percentage of proliferating (CFSE-low) (FIG.
10A, FIG. 10B, FIG. 10C), CD25 (FIG. 11A, FIG. 11B, FIG. 11C) and
CD44 (FIG. 12A, FIG. 12B, FIG. 12C) positive vital CFSE-labeled
murine CD3.sup.+CD8.sup.+ OT-1 T cells co-cultured with huCD40 tg
DCs pre-incubated with the indicated titrated antibodies in the
presence or absence of OVA. The x-axis shows the concentration of
antibody constructs.
[0171] FIG. 13A, FIG. 13B, FIG. 13C show IFN.gamma. levels measured
in the supernatants of T cells co-cultured with OVA-pulsed DCs
activated by FAP-targeted anti-CD40 binding molecules. IFN.gamma.
levels were elevated in conditions with T cells co-cultured with
DCs treated with the anti-human CD40 FAP-targeting antibody in the
presence of FAP (FIG. 13A). In addition, IFNy secretion was
increased to a similar extent with both, the anti-human CD40 and
the anti-mouse CD40 bispecific antigen binding molecules in a
FAP-dependent way. Shown is the IFN.gamma. amount in the
supernatant of murine CD3.sup.+CD8.sup.+ OT-1 T cells co-cultured
with huCD40 tg DCs pre-incubated with the indicated titrated
antibodies measured by ELISA. The x-axis shows the concentration of
antibody constructs.
[0172] FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG. 14F,
FIG. 14G, FIG. 14H show the in vitro activation of murine B cells
by mono- or bivalent FAP-targeted mouse anti-CD40 constructs in the
presence of FAP-coated or uncoated Dynabeads.RTM. after 2 days
incubation. With FAP-coated beads the bispecific antibody
monovalent for FAP induced a similar increase of B cell activation
marker expression (CD70, CD80, and CD86) as the bivalent
FAP-targeted molecule. Moreover, a significant B cell activation
marker upregulation was also observed for B cells treated with the
FAP-independent positive control antibody FGK4.5. In the absence of
FAP (uncoated beads) no increase of the B cell activation markers
CD70 and CD80 could be observed with the bispecific antigen binding
molecules. In contrast, the positive control antibody induced an
upregulation of CD70 and CD80 irrespective of FAP pre-treatment.
While CD86 upregulation was FAP-dependent with the tetravalent
anti-mouse CD40 antibody possessing two FAP binding moieties, a
FAP-independent effect was observed for the bispecific antigen
binding molecule having only one FAP binding site. In addition, a
FAP-independent upregulation of MHC-II expression was observed for
all tested bispecific antigen binding molecules. Shown is the
percentage of CD70 (FIG. 14A and FIG. 14B), CD80 (FIG. 14C and FIG.
14D), CD86 (FIG. 14E and FIG. 14F) and MHCII (FIG. 14G and FIG.
14H) positive vital B cells after 2 days incubation with the
indicated titrated antibodies. The x-axis shows the concentration
of antibody constructs.
[0173] FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, FIG. 15F,
FIG. 15G show schematic representations of the bispecific antigen
binding molecules which specifically bind to human CD40 and to FAP
or DP47. FIG. 15A shows a schematic representation of a bispecific
CD40-DP47 antibody in the 4+1 format consisting of four CD40
binding Fab domains combined with one DP47 binding moiety with VH
at the C-terminus of one heavy chain and VL at the C-terminus of
the other heavy chain (tetravalent for CD40 and monovalent for
DP47). The black point symbolizes knob-into-hole mutations. FIG.
15B shows a schematic representation of a bispecific CD40-DP47
antibody in the 4+2 format consisting of four CD40 binding Fab
domains combined with two DP47 binding Fab domains fused each at
the C-terminus of the heavy chains (tetravalent for CD40 and
bivalent for DP47). FIG. 15C shows a schematic representation of a
bispecific CD40-FAP antibody in the 1+1 format consisting of one
CD40 binding arm combined with one FAP binding arm (monovalent for
CD40 and monovalent for FAP). FIG. 15D shows a schematic scheme of
an exemplary bispecific CD40-FAP antibody in the 2+1 format
consisting of two CD40 binding Fab domains combined with one FAP
binding Fab domain as part of one of the two CD40 binding arms.
FIG. 15E shows a schematic representation of a bispecific CD40-FAP
antibody in the 4+1 format consisting of four CD40 binding Fab
domains combined with one FAP binding Fab domains fused at the
C-terminus of one of the heavy chains (tetravalent for CD40 and
monovalent for FAP). FIG. 15F shows a schematic representation of a
bispecific CD40-FAP antibody in the 4+1 format consisting of four
CD40 binding Fab domains combined with one FAP binding Fab domains
fused at the C-terminus of one of the heavy chains. The VH2a and
VL2a CD40 binding domains were obtained from an in-house
humanization of the murine S2C6 CD40 binding domain. FIG. 15G shows
a schematic representation of a bispecific CD40-FAP antibody in the
4+1 format consisting of four CD40 binding Fab domains combined
with one FAP binding Fab domains fused at the C-terminus of one of
the heavy chains. The VH2d and VL2a CD40 binding domains were
obtained from an in-house humanization of the murine S2C6 CD40
binding domain. FIG. 15H shows a schematic representation of a
bispecific CD40-FAP antibodies in the 2+1 format consisting of two
CD40 binding moieties combined with one FAP binding moiety as
crossover fab fragment, wherein the VL-CH1 chain is fused at the
C-terminus of the Fc knob chain. FIG. 15I shows a schematic
representation of a bispecific CD40-FAP antibodies in the 2+1
format consisting of two CD40 binding moieties combined with one
FAP binding moiety as crossover fab fragment, wherein the VH-CL
chain is fused at the C-terminus of the Fc knob chain.
[0174] FIG. 16A shows the parental murine FGK4.5 antibody
(P1AD3449). FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E show schematic
representations of the bispecific antigen binding molecules which
specifically bind to mouse CD40 and to FAP or DP47. FIG. 16B shows
a schematic representation of a bispecific CD40-FAP antibody in the
4+1 format consisting of four mouse CD40 binding Fab domains
combined with one FAP binding moiety with VH at the C-terminus of
one heavy chain and VL at the C-terminus of the other heavy chain
(tetravalent for CD40 and monovalent for FAP). The black point
symbolizes DD/KK mutations in the Fc and binding to Fc receptors is
inhibited by D270A/P329G mutations. FIG. 16C shows a schematic
representation of a bispecific CD40-FAP antibody in the 4+2 format
consisting of four mouse CD40 binding Fab domains combined with two
FAP binding Fab domains fused each at the C-terminus of the heavy
chains (tetravalent for CD40 and bivalent for FAP). Binding to Fc
receptors is inhibited by D270A/P329G mutations. FIG. 16D shows a
schematic representation of a bispecific CD40-DP47 antibody in the
4+1 format consisting of four mouse CD40 binding Fab domains
combined with one DP47 binding moiety with VH at the C-terminus of
one heavy chain and VL at the C-terminus of the other heavy chain
(tetravalent for CD40 and monovalent for DP47). The black point
symbolizes DD/KK mutations in the Fc and binding to Fc receptors is
inhibited by D270A/P329G mutations. FIG. 16E shows a schematic
representation of a bispecific CD40-DP47 antibody in the 4+2 format
consisting of four mouse CD40 binding Fab domains combined with two
DP47 binding Fab domains fused each at the C-terminus of the heavy
chains (tetravalent for CD40 and bivalent for DP47). Binding to Fc
receptors is inhibited by D270A/P329G mutations.
[0175] FIG. 17 shows the binding of human tetravalent, bivalent or
monovalent anti-CD40 antibodies in a FAP-targeted monovalent or
bivalent format to FAP positive tumor cells. The transgenic
modified mouse embryonic fibroblast NIH/3T3-mFAP cell line
expresses high levels of murine fibroblast activation protein
(mFAP). All depicted constructs vary in their binding strength
(EC.sub.50 values as well as signal strength) to NIH/3T3-mFAP
cells. Only the anti-CD40 antigen binding molecules with either one
or two FAP binding moieties but not the non-FAP-targeted formats
(P1AD4574 and P1AD4465) efficiently bind to NIH/3T3-mFAP cells. The
bivalent FAP constructs with C-terminal FAP binding domains bind
stronger than the monovalent construct with C-terminal FAP binding
domains. The strongest FAP binding was observed for the 1+1 format.
Shown is the binding as median of fluorescence intensity (MFI) of
phycoerythrin (PE)-labeled anti-human IgG Fc.gamma.-specific goat
IgG F(ab')2 fragment which is used as secondary detection antibody.
MFI was measured by flow cytometry. The x-axis shows the
concentration of antibody constructs.
[0176] FIG. 18 shows the binding of human tetravalent, bivalent or
monovalent anti-CD40 antibodies in a FAP-targeted monovalent or
bivalent format to Daudi cells, a B lymphoblast cell line with high
surface expression levels of human CD40. All depicted constructs
bind to CD40 but vary in their binding strength (EC.sub.50 values
as well as signal strength) to CD40-positive Daudi cells. Bivalent
anti-CD40 antibodies show higher EC.sub.50 levels and reach higher
binding plateaus compared to tetravalent anti-CD40 antibodies. The
highest EC.sub.50 value combined with the lowest binding plateau
was observed for the 1+1 format. Binding of anti-CD40 antibodies to
cell surface proteins was detected with an anti-human IgG
Fc.gamma.-specific goat IgG F(ab')2 fragment conjugated to
phycoerythrin (PE) using FACS analysis. MFI was measured by flow
cytometry and baseline corrected by subtracting the MFI of the
blank control. The x-axis shows the concentration of antibody
constructs.
[0177] FIG. 19A and FIG. 19B show the in vitro activation of
HEK-Blue.TM. CD40L cells by mono- or bivalent FAP-targeted human
anti-CD40 constructs in the presence of FAP-coated (FIG. 19A) or
uncoated Dynabeads.RTM. (FIG. 19B) after 24 hours incubation. In
the presence of FAP-coated beads the bispecific antibody monovalent
for FAP and mono- or bivalent for CD40 induced a similar increase
of SEAP production as the bispecific antibody bivalent for FAP and
CD40, whereas in the absence of FAP (uncoated beads) no or low SEAP
production was detected. Moreover, an upregulation of SEAP
production by FAP-targeted antibodies tetravalent for CD40 was
observed in the presence of FAP. However, SEAP production was also
observed in the absence of FAP in the supernatant of reporter cells
treated with FAP-targeted antibodies tetravalent for CD40. The
negative control antibody tetravalent for human CD40 with one DP47
domain instead of a FAP binding domain induced comparable SEAP
production in HEK-Blue.TM. CD40L cells in the presence and absence
of FAP and the positive control antibody P1AD4470+F(ab) induced
similar levels of SEAP production as compared to FAP-targeted
bispecific antibodies bivalent or tetravalent for human CD40 in the
presence of FAP-coated beads. Shown is the absorption at a
wavelength of 650 nm which correlates with the amount of hydrolyzed
substrate by SEAP. The x-axis shows the concentration of antibody
constructs. The EC.sub.50 values of HEK-Blue.TM. CD40L cell
activation in the presence of FAP-coated beads are summarized in
Table 11. The EC.sub.50 values of all tested antibodies tetravalent
for CD40 were comparable and lower compared to the EC.sub.50 values
of the depicted antibodies bivalent for CD40. The highest EC.sub.50
value was detected for the 1+1 format.
[0178] FIG. 20A and FIG. 20B show the in vitro activation of human
Daudi cells by mono- or bivalent FAP-targeted human anti-CD40
constructs in the presence of FAP-coated (FIG. 20A) or uncoated
Dynabeads.RTM. (FIG. 20B) after 2 days incubation. With FAP-coated
beads the bispecific antibodies monovalent for FAP induced a
similar increase of the B cell activation marker expression CD70 as
the bivalent FAP-targeted molecules. Moreover, the B cell
activation marker upregulation by FAP-targeted bispecific
antigen-binding molecules was higher comparable to the upregulation
induced by the FAP-independent positive control antibodies. In the
absence of FAP (uncoated beads) no increase of CD70 could be
observed with the depicted FAP-targeted bispecific antibodies mono-
or bivalent for CD40, while tetravalent CD40 binding molecules
induce an upregulation of CD70, but to a lesser extent than in the
presence of FAP. Shown is the percentage of CD70 positive vital
Daudi cells after 2 days incubation with the indicated titrated
antibodies. The x-axis shows the concentration of antibody
constructs. The EC.sub.50 values of activation in the presence of
FAP-coated beads are summarized in Table 9. The EC.sub.50 values of
all FAP-targeted antibodies tetravalent for CD40 were comparable
and lower compared to the EC.sub.50 values of the depicted
antibodies bivalent for CD40. The highest EC.sub.50 values were
detected for the positive control antibody P1AD4470 and the 1+1
format.
[0179] FIG. 21A and FIG. 21B show the in vitro activation of human
B cells by mono- or bivalent FAP-targeted human anti-CD40
constructs in the presence of FAP-coated (FIG. 21A) or uncoated
Dynabeads.RTM. (FIG. 21B) after 2 days incubation. With FAP-coated
beads the bispecific antibodies monovalent for FAP induced a
similar increase of the B cell activation marker expression CD86 as
the bivalent FAP-targeted molecules. Compared to the
FAP-independent upregulation of CD86 induced by cross-linked CD40
antibody (P1AD4470), CD86 upregulation induced by FAP-dependent
bispecific antigen binding molecules was slightly lower. In the
absence of FAP (uncoated beads) no increase of CD86 expression
could be observed with the bispecific antigen binding molecules,
while positive control antibodies induced an upregulation of
activation markers. Shown is the percentage of CD86 positive vital
B cells after 2 days incubation with the indicated titrated
antibodies. The x-axis shows the concentration of antibody
constructs. The EC.sub.50 values of activation in the presence of
FAP-coated beads are summarized in Table 10. The EC.sub.50 values
of all FAP-targeted antibodies tetravalent for CD40 were comparable
and lower compared to the EC.sub.50 values of the depicted
FAP-targeted antibodies bivalent for CD40. The highest EC.sub.50
values were detected for the 2+1, 2+2, and 1+1 format.
[0180] FIG. 22A and FIG. 22B show the T cell priming of OVA-pulsed
DCs activated by FAP-targeted anti-CD40 binding molecules in the
presence (FIG. 22A) or absence (FIG. 22B) of FAP. DCs isolated from
huCD40 transgenic mice, treated with DEC205-OVA conjugate and
stimulated with FAP-dependent bispecific anti-CD40 antibodies as
well as FAP-coated beads induced a strong proliferation of
antigen-specific T cells. In contrast, in the absence of FAP
(uncoated beads) no or little T cell proliferation and activation
was induced by DCs stimulated with FAP-targeted anti-CD40
antibodies. T cell proliferation induced by DCs stimulated with the
human bispecific antigen binding molecules with one, two or four
CD40 and one or two FAP binding moieties was comparable. DCs pulsed
with high amounts of SIINFEKL instead of DEC205-OVA conjugate
induced a strong T cell proliferation. Shown is the percentage of
proliferating (CFSE-low) vital CFSE-labeled murine
CD3.sup.+CD8.sup.+ OT-1 T cells co-cultured with huCD40 tg DCs
pre-incubated with the indicated titrated antibodies in the
presence of OVA (FIG. 22A and FIG. 22B). The x-axis shows the
concentration of antibody constructs.
[0181] FIG. 23A, FIG. 23B, FIG. 23C, FIG. 23D, and FIG. 23E; and
FIG. 24A, FIG. 24B, FIG. 24C, and FIG. 24D show enzyme serum
levels, body weight, spleen weight, DC activation and T cell
proliferation in mice injected with a FAP-expressing murine colon
adenocarcinoma tumor cell line (MC38-FAP) and treated with either
FGK4.5 (PlAD3449) or FGK4.5.times.FAP 4+1 (P1AD4520) or vehicle
alone. In contrast to mice treated with non-targeted CD40 mAb
(FGK4.5), treatment with FAP-CD40 (FGK4.5) 4+1, (i.e a bispecific
antibody tetravalent for CD40 and monovalent for FAP) did not
induce liver injury as no increase in serum enzymes indicative of
liver injury was observed. This is shown for 3 animals per group in
FIG. 23A for alanine aminotransferase (ALT), in FIG. 23B for
glutamate dehydrogenase (GDH) and in FIG. 23C for sorbitol
dehydrogenase (SDH). Moreover, no decrease in body weight (FIG.
23D) and less increase in spleen weight (FIG. 23E) was observed in
mice treated with FGK4.5.times.FAP (P1AD4520) compared to mice
treated with the parental untargeted CD40 antibody (P1AD3449). DC
activation in the tumor-draining lymph nodes three days post
therapy injection (FIG. 24A and FIG. 24B) and T cell proliferation
in tumor eight days post therapy injection (FIG. 24C and FIG. 24D)
was significantly increased in FGK4.5- and FGK4.5.times.FAP
4+1-treated animals compared to vehicle-treated animals. In FIG.
23A, FIG. 23B, and FIG. 23C the y-axis shows serum enzyme levels in
units per liter and the x-axis shows individual mice treated with
FGK4.5, FGK4.5.times.FAP 4+1 or vehicle alone. In FIG. 23D the
y-axis shows the body weight in gram of mice treated FGK4.5,
FGK4.5.times.FAP 4+1 or vehicle alone and the x-axis shows the days
post tumor injection. FIG. 23E shows the spleen weight of mice
treated with FGK4.5, FGK4.5.times.FAP 4+1 or vehicle alone three
days post therapy injection. FIG. 24A, FIG. 24B, FIG. 24C, and FIG.
24D show the CD86 (FIG. 24A) and CD70 expression (FIG. 24B) of DCs
in the tumor-draining lymph node and the Ki67 expression of
CD8.sup.+ T cells (FIG. 24C) and the total numbers of CD8.sup.+ T
cell (FIG. 24D) in the tumor three and eight days post therapy
injection, respectively. ((*p<0.05, **p<0.01, ***p<0.001,
unpaired, two-tailed Student's test).
[0182] FIG. 25A and FIG. 25B show the in vitro activation of human
B cells by bivalent FAP-targeted human anti-CD40 constructs in the
presence of FAP-coated (FIG. 25A and FIG. 25C) or uncoated
Dynabeads.RTM. (FIG. 25B and FIG. 25D) after 2 days incubation.
Compared to the FAP-independent upregulation of CD70 and CD86
induced by the cross-linked CD40 antibody (P1AA5145), CD70
upregulation (FIG. 25A) and CD86 upregulation (FIG. 25C) induced by
FAP-dependent bispecific antigen binding molecules was slightly
lower. In the absence of FAP (uncoated beads) no increase of CD70
(FIG. 25B) or CD86 expression (FIG. 25D) could be observed with the
bispecific antigen binding molecules, while positive control
antibodies induced an upregulation of activation markers. Shown is
the percentage of CD70 or CD86 positive vital B cells after 2 days
incubation with the indicated titrated antibodies. The x-axis shows
the concentration of antibody constructs.
DETAILED DESCRIPTION OF THE INVENTION
[0183] Definitions
[0184] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as generally used in the art to
which this invention belongs. For purposes of interpreting this
specification, the following definitions will apply and whenever
appropriate, terms used in the singular will also include the
plural and vice versa.
[0185] As used herein, the term "antigen binding molecule" refers
in its broadest sense to a molecule that specifically binds an
antigenic determinant. Examples of antigen binding molecules are
antibodies, antibody fragments and scaffold antigen binding
proteins.
[0186] As used herein, the term "antigen binding domain capable of
specific binding to a target cell antigen" or "moiety capable of
specific binding to a target cell antigen" refers to a polypeptide
molecule that specifically binds to an antigenic determinant. In
one aspect, the antigen binding domain is able to activate
signaling through its target cell antigen. In a particular aspect,
the antigen binding domain is able to direct the entity to which it
is attached (e.g. the CD40 agonist) to a target site, for example
to a specific type of tumor cell or tumor stroma bearing the
antigenic determinant. Antigen binding domains capable of specific
binding to a target cell antigen include antibodies and fragments
thereof as further defined herein. In addition, antigen binding
domains capable of specific binding to a target cell antigen
include scaffold antigen binding proteins as further defined
herein, e.g. binding domains which are based on designed repeat
proteins or designed repeat domains (see e.g. WO 2002/020565).
[0187] In relation to an antibody or fragment thereof, the term
"antigen binding domain capable of specific binding to a target
cell antigen" refers to the part of the molecule that comprises the
area which specifically binds to and is complementary to part or
all of an antigen. A antigen binding domain capable of specific
antigen binding may be provided, for example, by one or more
antibody variable domains (also called antibody variable regions).
Particularly, an antigen binding domain capable of specific antigen
binding comprises an antibody light chain variable region (VL) and
an antibody heavy chain variable region (VH). In another aspect,
the "antigen binding domain capable of specific binding to a target
cell antigen " can also be a Fab fragment or a cross-Fab
fragment.
[0188] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, monospecific and
multispecific antibodies (e.g., bispecific antibodies), and
antibody fragments so long as they exhibit the desired
antigen-binding activity.
[0189] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g. containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen.
[0190] The term "monospecific" antibody as used herein denotes an
antibody that has one or more binding sites each of which bind to
the same epitope of the same antigen. The term "bispecific" means
that the antigen binding molecule is able to specifically bind to
at least two distinct antigenic determinants. Typically, a
bispecific antigen binding molecule comprises two antigen binding
sites, each of which is specific for a different antigenic
determinant. In certain embodiments the bispecific antigen binding
molecule is capable of simultaneously binding two antigenic
determinants, particularly two antigenic determinants expressed on
two distinct cells. A bispecific antigen binding molecule as
described herein can also form part of a multispecific
antibody.
[0191] The term "valent" as used within the current application
denotes the presence of a specified number of binding sites
specific for one distinct antigenic determinant in an antigen
binding molecule that are specific for one distinct antigenic
determinant. As such, the terms "bivalent", "tetravalent", and
"hexavalent" denote the presence of two binding sites, four binding
sites, and six binding sites specific for a certain antigenic
determinant, respectively, in an antigen binding molecule. In
particular aspects of the invention, the bispecific antigen binding
molecules according to the invention can be monovalent for a
certain antigenic determinant, meaning that they have only one
binding site for said antigenic determinant or they can be bivalent
or tetravalent for a certain antigenic determinant, meaning that
they have two binding sites or four binding sites, respectively,
for said antigenic determinant.
[0192] The terms "full length antibody", "intact antibody", and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure. "Native antibodies" refer to naturally
occurring immunoglobulin molecules with varying structures. For
example, native IgG-class antibodies are heterotetrameric
glycoproteins of about 150,000 daltons, composed of two light
chains and two heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3), also called
a heavy chain constant region. Similarly, from N- to C-terminus,
each light chain has a variable region (VL), also called a variable
light domain or a light chain variable domain, followed by a light
chain constant domain (CL), also called a light chain constant
region. The heavy chain of an antibody may be assigned to one of
five types, called .alpha. (IgA), .delta. (IgD), .epsilon. (IgE),
.gamma. (IgG), or .mu. (IgM), some of which may be further divided
into subtypes, e.g. .gamma.1 (IgG1), .gamma.2 (IgG2), .gamma.3
(IgG3), .gamma.4 (IgG4), .alpha.1 (IgA1) and .alpha.2 (IgA2). The
light chain of an antibody may be assigned to one of two types,
called kappa (.kappa.) and lambda (.lamda.), based on the amino
acid sequence of its constant domain.
[0193] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies, triabodies, tetrabodies,
cross-Fab fragments; linear antibodies; single-chain antibody
molecules (e.g. scFv); and single domain antibodies. For a review
of certain antibody fragments, see Hudson et al., Nat Med 9,
129-134 (2003). For a review of scFv fragments, see e.g. Pluckthun,
in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg
and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see
also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab')2 fragments comprising salvage receptor
binding epitope residues and having increased in vivo half-life,
see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with
two antigen-binding sites that may be bivalent or bispecific, see,
for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9,
129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90,
6444-6448 (1993). Triabodies and tetrabodies are also described in
Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies
are antibody fragments comprising all or a portion of the heavy
chain variable domain or all or a portion of the light chain
variable domain of an antibody. In certain embodiments, a
single-domain antibody is a human single-domain antibody (Domantis,
Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1).
Antibody fragments can be made by various techniques, including but
not limited to proteolytic digestion of an intact antibody as well
as production by recombinant host cells (e.g. E. coli or phage), as
described herein.
[0194] Papain digestion of intact antibodies produces two identical
antigen-binding fragments, called "Fab" fragments containing each
the heavy- and light-chain variable domains and also the constant
domain of the light chain and the first constant domain (CH1) of
the heavy chain. As used herein, Thus, the term "Fab fragment"
refers to an antibody fragment comprising a light chain fragment
comprising a VL domain and a constant domain of a light chain (CL),
and a VH domain and a first constant domain (CH1) of a heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteins from the antibody hinge region.
Fab'-SH are Fab' fragments wherein the cysteine residue(s) of the
constant domains bear a free thiol group. Pepsin treatment yields
an F(ab')2 fragment that has two antigen-combining sites (two Fab
fragments) and a part of the Fc region. According to the present
invention, the term "Fab fragment" also includes "cross-Fab
fragments" or "crossover Fab fragments" as defined below.
[0195] The term "cross-Fab fragment" or "xFab fragment" or
"crossover Fab fragment" refers to a Fab fragment, wherein either
the variable regions or the constant regions of the heavy and light
chain are exchanged. Two different chain compositions of a
crossover Fab molecule are possible and comprised in the bispecific
antibodies of the invention: On the one hand, the variable regions
of the Fab heavy and light chain are exchanged, i.e. the crossover
Fab molecule comprises a peptide chain composed of the light chain
variable region (VL) and the heavy chain constant region (CH1), and
a peptide chain composed of the heavy chain variable region (VH)
and the light chain constant region (CL). This crossover Fab
molecule is also referred to as CrossFab.sub.(VLVH). On the other
hand, when the constant regions of the Fab heavy and light chain
are exchanged, the crossover Fab molecule comprises a peptide chain
composed of the heavy chain variable region (VH) and the light
chain constant region (CL), and a peptide chain composed of the
light chain variable region (VL) and the heavy chain constant
region (CH1). This crossover Fab molecule is also referred to as
CrossFab.sub.(CLCH1).
[0196] A "single chain Fab fragment" or "scFab" is a polypeptide
consisting of an antibody heavy chain variable domain (VH), an
antibody constant domain 1 (CH1), an antibody light chain variable
domain (VL), an antibody light chain constant domain (CL) and a
linker, wherein said antibody domains and said linker have one of
the following orders in N-terminal to C-terminal direction: a)
VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1
or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide
of at least 30 amino acids, preferably between 32 and 50 amino
acids. Said single chain Fab fragments are stabilized via the
natural disulfide bond between the CL domain and the CH1 domain. In
addition, these single chain Fab molecules might be further
stabilized by generation of interchain disulfide bonds via
insertion of cysteine residues (e.g. position 44 in the variable
heavy chain and position 100 in the variable light chain according
to Kabat numbering).
[0197] A "crossover single chain Fab fragment" or "x-scFab" is a is
a polypeptide consisting of an antibody heavy chain variable domain
(VH), an antibody constant domain 1 (CH1), an antibody light chain
variable domain (VL), an antibody light chain constant domain (CL)
and a linker, wherein said antibody domains and said linker have
one of the following orders in N-terminal to C-terminal direction:
a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and
VL form together an antigen-binding site which binds specifically
to an antigen and wherein said linker is a polypeptide of at least
30 amino acids. In addition, these x-scFab molecules might be
further stabilized by generation of interchain disulfide bonds via
insertion of cysteine residues (e.g. position 44 in the variable
heavy chain and position 100 in the variable light chain according
to Kabat numbering).
[0198] A "single-chain variable fragment (scFv)" is a fusion
protein of the variable regions of the heavy (V.sub.H) and light
chains (V.sub.L) of an antibody, connected with a short linker
peptide of ten to about 25 amino acids. The linker is usually rich
in glycine for flexibility, as well as serine or threonine for
solubility, and can either connect the N-terminus of the V.sub.H
with the C-terminus of the V.sub.L, or vice versa. This protein
retains the specificity of the original antibody, despite removal
of the constant regions and the introduction of the linker. scFv
antibodies are, e.g. described in Houston, J. S., Methods in
Enzymol. 203 (1991) 46-96). In addition, antibody fragments
comprise single chain polypeptides having the characteristics of a
VH domain, namely being able to assemble together with a VL domain,
or of a VL domain, namely being able to assemble together with a VH
domain to a functional antigen binding site and thereby providing
the antigen binding property of full length antibodies.
[0199] "Scaffold antigen binding proteins" are known in the art,
for example, fibronectin and designed ankyrin repeat proteins
(DARPins) have been used as alternative scaffolds for
antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered
protein scaffolds as next-generation antibody therapeutics. Curr
Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new
generation of protein therapeutics. Drug Discovery Today 13:
695-701 (2008). In one aspect of the invention, a scaffold antigen
binding protein is selected from the group consisting of CTLA-4
(Evibody), Lipocalins (Anticalin), a Protein A-derived molecule
such as Z-domain of Protein A (Affibody), an A-domain
(Avimer/Maxibody), a serum transferrin (trans-body); a designed
ankyrin repeat protein (DARPin), a variable domain of antibody
light chain or heavy chain (single-domain antibody, sdAb), a
variable domain of antibody heavy chain (nanobody, aVH), V.sub.NAR
fragments, a fibronectin (AdNectin), a C-type lectin domain
(Tetranectin); a variable domain of a new antigen receptor
beta-lactamase (V.sub.NAR fragments), a human gamma-crystallin or
ubiquitin (Affilin molecules); a kunitz type domain of human
protease inhibitors, microbodies such as the proteins from the
knottin family, peptide aptamers and fibronectin (adnectin). CTLA-4
(Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family
receptor expressed on mainly CD4.sup.+ T-cells. Its extracellular
domain has a variable domain-like Ig fold. Loops corresponding to
CDRs of antibodies can be substituted with heterologous sequence to
confer different binding properties. CTLA-4 molecules engineered to
have different binding specificities are also known as Evibodies
(e.g. U.S. Pat. No. 7,166,697B1). Evibodies are around the same
size as the isolated variable region of an antibody (e.g. a domain
antibody). For further details see Journal of Immunological Methods
248 (1-2), 31-45 (2001). Lipocalins are a family of extracellular
proteins which transport small hydrophobic molecules such as
steroids, bilins, retinoids and lipids. They have a rigid
beta-sheet secondary structure with a number of loops at the open
end of the conical structure which can be engineered to bind to
different target antigens. Anticalins are between 160-180 amino
acids in size, and are derived from lipocalins. For further details
see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No.
7,250,297B1 and US20070224633. An affibody is a scaffold derived
from Protein A of Staphylococcus aureus which can be engineered to
bind to antigen. The domain consists of a three-helical bundle of
approximately 58 amino acids. Libraries have been generated by
randomization of surface residues. For further details see Protein
Eng. Des. Sel. 2004, 17, 455-462 and EP 1641818A1. Avimers are
multidomain proteins derived from the A-domain scaffold family. The
native domains of approximately 35 amino acids adopt a defined
disulfide bonded structure. Diversity is generated by shuffling of
the natural variation exhibited by the family of A-domains. For
further details see Nature Biotechnology 23(12), 1556-1561 (2005)
and Expert Opinion on Investigational Drugs 16(6), 909-917 (June
2007). A transferrin is a monomeric serum transport glycoprotein.
Transferrins can be engineered to bind different target antigens by
insertion of peptide sequences in a permissive surface loop.
Examples of engineered transferrin scaffolds include the
Trans-body. For further details see J. Biol. Chem 274, 24066-24073
(1999). Designed Ankyrin Repeat Proteins (DARPins) are derived from
Ankyrin which is a family of proteins that mediate attachment of
integral membrane proteins to the cytoskeleton. A single ankyrin
repeat is a 33 residue motif consisting of two alpha-helices and a
beta-turn. They can be engineered to bind different target antigens
by randomizing residues in the first alpha-helix and a beta-turn of
each repeat. Their binding interface can be increased by increasing
the number of modules (a method of affinity maturation). For
further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4),
1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and
US20040132028A1. A single-domain antibody is an antibody fragment
consisting of a single monomeric variable antibody domain. The
first single domains were derived from the variable domain of the
antibody heavy chain from camelids (nanobodies or V.sub.HH
fragments). Furthermore, the term single-domain antibody includes
an autonomous human heavy chain variable domain (aVH) or V.sub.NAR
fragments derived from sharks. Fibronectin is a scaffold which can
be engineered to bind to antigen. Adnectins consists of a backbone
of the natural amino acid sequence of the 10th domain of the 15
repeating units of human fibronectin type III (FN3). Three loops at
one end of the .beta.-sandwich can be engineered to enable an
Adnectin to specifically recognize a therapeutic target of
interest. For further details see Protein Eng. Des. Sel. 18,
435-444 (2005), US20080139791, WO2005056764 and U.S. Pat. No.
6,818,418B1. Peptide aptamers are combinatorial recognition
molecules that consist of a constant scaffold protein, typically
thioredoxin (TrxA) which contains a constrained variable peptide
loop inserted at the active site. For further details see Expert
Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from
naturally occurring microproteins of 25-50 amino acids in length
which contain 3-4 cysteine bridges--examples of microproteins
include KalataBI and conotoxin and knottins. The microproteins have
a loop which can beengineered to include upto 25 amino acids
without affecting the overall fold of the microprotein. For further
details of engineered knottin domains, see WO2008098796.
[0200] An "antigen binding molecule that binds to the same epitope"
as a reference molecule refers to an antigen binding molecule that
blocks binding of the reference molecule to its antigen in a
competition assay by 50% or more, and conversely, the reference
molecule blocks binding of the antigen binding molecule to its
antigen in a competition assay by 50% or more.
[0201] The term "antigen binding domain" or "antigen-binding site"
refers to the part of an antigen binding molecule that comprises
the area which specifically binds to and is complementary to part
or all of an antigen. Where an antigen is large, an antigen binding
molecule may only bind to a particular part of the antigen, which
part is termed an epitope. An antigen binding domain may be
provided by, for example, one or more variable domains (also called
variable regions). Preferably, an antigen binding domain comprises
an antibody light chain variable region (VL) and an antibody heavy
chain variable region (VH).
[0202] As used herein, the term "antigenic determinant" is
synonymous with "antigen" and "epitope," and refers to a site (e.g.
a contiguous stretch of amino acids or a conformational
configuration made up of different regions of non-contiguous amino
acids) on a polypeptide macromolecule to which an antigen binding
moiety binds, forming an antigen binding moiety-antigen complex.
Useful antigenic determinants can be found, for example, on the
surfaces of tumor cells, on the surfaces of virus-infected cells,
on the surfaces of other diseased cells, on the surface of immune
cells, free in blood serum, and/or in the extracellular matrix
(ECM). The proteins useful as antigens herein can be any native
form the proteins from any vertebrate source, including mammals
such as primates (e.g. humans) and rodents (e.g. mice and rats),
unless otherwise indicated. In a particular embodiment the antigen
is a human protein. Where reference is made to a specific protein
herein, the term encompasses the "full-length", unprocessed protein
as well as any form of the protein that results from processing in
the cell. The term also encompasses naturally occurring variants of
the protein, e.g. splice variants or allelic variants.
[0203] By "specific binding" is meant that the binding is selective
for the antigen and can be discriminated from unwanted or
non-specific interactions. The ability of an antigen binding
molecule to bind to a specific antigen can be measured either
through an enzyme-linked immunosorbent assay (ELISA) or other
techniques familiar to one of skill in the art, e.g. Surface
Plasmon Resonance (SPR) technique (analyzed on a BlAcore
instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and
traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
In one embodiment, the extent of binding of an antigen binding
molecule to an unrelated protein is less than about 10% of the
binding of the antigen binding molecule to the antigen as measured,
e.g. by SPR. In certain embodiments, an molecule that binds to the
antigen has a dissociation constant (Kd) of .ltoreq.1.mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8 M or less,
e.g. from 10.sup.-8 M to 10.sup.-13 M, e.g. from 10.sup.-9 M to
10.sup.-13 M).
[0204] "Affinity" or "binding affinity" refers to the strength of
the sum total of non-covalent interactions between a single binding
site of a molecule (e.g. an antibody) and its binding partner (e.g.
an antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g.
[0205] antibody and antigen). The affinity of a molecule X for its
partner Y can generally be represented by the dissociation constant
(Kd), which is the ratio of dissociation and association rate
constants (koff and kon, respectively). Thus, equivalent affinities
may comprise different rate constants, as long as the ratio of the
rate constants remains the same. Affinity can be measured by common
methods known in the art, including those described herein. A
particular method for measuring affinity is Surface Plasmon
Resonance (SPR).
[0206] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0207] A "target cell antigen" as used herein refers to an
antigenic determinant presented on the surface of a target cell, in
particular a target cell in a tumor such as a cancer cell or a cell
of the tumor stroma. Thus, the target cell antigen is a
tumor-associated antigen. In particular, a target cell antigen does
not include immune checkpoint receptors on activated T cells, such
as CTLA-4, PD-1 or PD-L1. In certain embodiments, the target cell
antigen is an antigen on the surface of a tumor cell. In one
aspect, the tumor target cell antigen is selected from the group
consisting of Fibroblast Activation Protein (FAP), Carcinoembryonic
Antigen (CEA), Melanoma-associated Chondroitin Sulfate Proteoglycan
(MCSP), Epidermal Growth Factor Receptor (EGFR), CD19, CD20 and
CD33. In particular, the tumor target cell antigen is Fibroblast
Activation Protein (FAP).
[0208] The term "Fibroblast activation protein (FAP)", also known
as Prolyl endopeptidase FAP or Seprase (EC 3.4.21), refers to any
native FAP from any vertebrate source, including mammals such as
primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys)
and rodents (e.g. mice and rats), unless otherwise indicated. The
term encompasses "full-length," unprocessed FAP as well as any form
of FAP that results from processing in the cell. The term also
encompasses naturally occurring variants of FAP, e.g., splice
variants or allelic variants. In one embodiment, the antigen
binding molecule of the invention is capable of specific binding to
human, mouse and/or cynomolgus FAP. The amino acid sequence of
human FAP is shown in UniProt (www.uniprot.org) accession no.
Q12884 (version 149, SEQ ID NO:2), or NCBI (www.ncbi.nlm.nih.gov/)
RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP
extends from amino acid position 26 to 760. The amino acid sequence
of a His-tagged human FAP ECD is shown in SEQ ID NOs 142. The amino
acid sequence of mouse FAP is shown in UniProt accession no. P97321
(version 126, SEQ ID NO:143), or NCBI RefSeq NP_032012.1. The
extracellular domain (ECD) of mouse FAP extends from amino acid
position 26 to 761. SEQ ID NO: 144 shows the amino acid of a
His-tagged mouse FAP ECD. SEQ ID NO:145 shows the amino acid of a
His-tagged cynomolgus FAP ECD. Preferably, an anti-FAP binding
molecule of the invention binds to the extracellular domain of
FAP.
[0209] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antigen binding molecule to antigen. The variable
domains of the heavy chain and light chain (VH and VL,
respectively) of a native antibody generally have similar
structures, with each domain comprising four conserved framework
regions (FRs) and three hypervariable regions (HVRs). See, e.g.,
Kindt et al., Kuby Immunology, 6th ed., W. H. Freeman and Co., page
91 (2007). A single VH or VL domain may be sufficient to confer
antigen-binding specificity.
[0210] The term "hypervariable region" or "HVR," as used herein
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
"complementarity determining regions" (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. Exemplary hypervariable loops occur at amino acid
residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55
(H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,
and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2,
89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991).) Hypervariable regions (HVRs) are also referred to as
complementarity determining regions (CDRs), and these terms are
used herein interchangeably in reference to portions of the
variable region that form the antigen binding regions. This
particular region has been described by Kabat et al., U.S. Dept. of
Health and Human Services, "Sequences of Proteins of Immunological
Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917
(1987), where the definitions include overlapping or subsets of
amino acid residues when compared against each other. Nevertheless,
application of either definition to refer to a CDR of an antibody
or variants thereof is intended to be within the scope of the term
as defined and used herein. The appropriate amino acid residues
which encompass the CDRs as defined by each of the above cited
references are set forth below in Table A as a comparison. The
exact residue numbers which encompass a particular CDR will vary
depending on the sequence and size of the CDR. Those skilled in the
art can routinely determine which residues comprise a particular
CDR given the variable region amino acid sequence of the
antibody.
TABLE-US-00001 TABLE A CDR Definitions.sup.1 CDR Kabat Chothia
AbM.sup.2 V.sub.H CDR1 31-35 26-32 26-35 V.sub.H CDR2 50-65 52-58
50-58 V.sub.H CDR3 95-102 95-102 95-102 V.sub.L CDR1 24-34 26-32
24-34 V.sub.L CDR2 50-56 50-52 50-56 V.sub.L CDR3 89-97 91-96 89-97
.sup.1Numbering of all CDR definitions in Table A is according to
the numbering conventions set forth by Kabat et al. (see below).
.sup.2"AbM" with a lowercase "b" as used in Table A refers to the
CDRs as defined by Oxford Molecular's "AbM" antibody modeling
software.
[0211] Kabat et al. also defined a numbering system for variable
region sequences that is applicable to any antibody. One of
ordinary skill in the art can unambiguously assign this system of
"Kabat numbering" to any variable region sequence, without reliance
on any experimental data beyond the sequence itself. As used
herein, "Kabat numbering" refers to the numbering system set forth
by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence
of Proteins of Immunological Interest" (1983). Unless otherwise
specified, references to the numbering of specific amino acid
residue positions in an antibody variable region are according to
the Kabat numbering system.
[0212] With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. CDRs
also comprise "specificity determining residues," or "SDRs," which
are residues that contact antigen. SDRs are contained within
regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary
a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2,
89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See
Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless
otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
[0213] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0214] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0215] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g. IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu. respectively.
[0216] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization. Other forms of "humanized antibodies" encompassed by
the present invention are those in which the constant region has
been additionally modified or changed from that of the original
antibody to generate the properties according to the invention,
especially in regard to C1q binding and/or Fc receptor (FcR)
binding.
[0217] A "human" antibody is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0218] The term "Fc domain" or "Fc region" herein is used to define
a C-terminal region of an antibody heavy chain that contains at
least a portion of the constant region. The term includes native
sequence Fc regions and variant Fc regions. An IgG Fc region
comprises an IgG CH2 and an IgG CH3 domain. The "CH2 domain" of a
human IgG Fc region usually extends from an amino acid residue at
about position 231 to an amino acid residue at about position 340.
In one embodiment, a carbohydrate chain is attached to the CH2
domain. The CH2 domain herein may be a native sequence CH2 domain
or variant CH2 domain. The "CH3 domain" comprises the stretch of
residues C-terminal to a CH2 domain in an Fc region (i.e. from an
amino acid residue at about position 341 to an amino acid residue
at about position 447 of an IgG). The CH3 region herein may be a
native sequence CH3 domain or a variant CH3 domain (e.g. a CH3
domain with an introduced "protuberance" ("knob") in one chain
thereof and a corresponding introduced "cavity" ("hole") in the
other chain thereof; see U.S. Pat. No. 5,821,333, expressly
incorporated herein by reference). Such variant CH3 domains may be
used to promote heterodimerization of two non-identical antibody
heavy chains as herein described. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0219] The "knob-into-hole" technology is described e.g. in U.S.
Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot
Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
Generally, the method involves introducing a protuberance ("knob")
at the interface of a first polypeptide and a corresponding cavity
("hole") in the interface of a second polypeptide, such that the
protuberance can be positioned in the cavity so as to promote
heterodimer formation and hinder homodimer formation. Protuberances
are constructed by replacing small amino acid side chains from the
interface of the first polypeptide with larger side chains (e.g.
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino acid side chains
with smaller ones (e.g. alanine or threonine). The protuberance and
cavity can be made by altering the nucleic acid encoding the
polypeptides, e.g. by site-specific mutagenesis, or by peptide
synthesis. In a specific embodiment a knob modification comprises
the amino acid substitution T366W in one of the two subunits of the
Fc domain, and the hole modification comprises the amino acid
substitutions T366S, L368A and Y407V in the other one of the two
subunits of the Fc domain. In a further specific embodiment, the
subunit of the Fc domain comprising the knob modification
additionally comprises the amino acid substitution S354C, and the
subunit of the Fc domain comprising the hole modification
additionally comprises the amino acid substitution Y349C.
Introduction of these two cysteine residues results in the
formation of a disulfide bridge between the two subunits of the Fc
region, thus further stabilizing the dimer (Carter, J Immunol
Methods 248, 7-15 (2001)).
[0220] A "region equivalent to the Fc region of an immunoglobulin"
is intended to include naturally occurring allelic variants of the
Fc region of an immunoglobulin as well as variants having
alterations which produce substitutions, additions, or deletions
but which do not decrease substantially the ability of the
immunoglobulin to mediate effector functions (such as
antibody-dependent cellular cytotoxicity). For example, one or more
amino acids can be deleted from the N-terminus or C-terminus of the
Fc region of an immunoglobulin without substantial loss of
biological function. Such variants can be selected according to
general rules known in the art so as to have minimal effect on
activity (see, e.g., Bowie, J. U. et al., Science 247:1306-10
(1990)).
[0221] The term "effector function" refers to those biological
activities attributable to the Fc region of an antibody, which vary
with the antibody isotype. Examples of antibody effector functions
include: C1q binding and complement dependent cytotoxicity (CDC),
Fc receptor binding, antibody-dependent cell-mediated cytotoxicity
(ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine
secretion, immune complex-mediated antigen uptake by antigen
presenting cells, down regulation of cell surface receptors (e.g. B
cell receptor), and B cell activation.
[0222] Fc receptor binding dependent effector functions can be
mediated by the interaction of the Fc-region of an antibody with Fc
receptors (FcRs), which are specialized cell surface receptors on
hematopoietic cells. Fc receptors belong to the immunoglobulin
superfamily, and have been shown to mediate both the removal of
antibody-coated pathogens by phagocytosis of immune complexes, and
the lysis of erythrocytes and various other cellular targets (e.g.
tumor cells) coated with the corresponding antibody, via antibody
dependent cell mediated cytotoxicity (ADCC) (see e.g. Van de
Winkel, J. G. and Anderson, C. L., J. Leukoc. Biol. 49 (1991)
511-524). FcRs are defined by their specificity for immunoglobulin
isotypes: Fc receptors for IgG antibodies are referred to as
Fc.gamma.R. Fc receptor binding is described e.g. in Ravetch, J. V.
and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P.
J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J.
Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E., et al.,
Ann. Hematol. 76 (1998) 231-248.
[0223] Cross-linking of receptors for the Fc-region of IgG
antibodies (Fc.gamma.R) triggers a wide variety of effector
functions including phagocytosis, antibody-dependent cellular
cytotoxicity, and release of inflammatory mediators, as well as
immune complex clearance and regulation of antibody production. In
humans, three classes of Fc.gamma.R have been characterized, which
are:
[0224] Fc.gamma.RI (CD64) binds monomeric IgG with high affinity
and is expressed on macrophages, monocytes, neutrophils and
eosinophils. Modification in the Fc-region IgG at least at one of
the amino acid residues E233-G236, P238, D265, N297, A327 and P329
(numbering according to EU index of Kabat) reduce binding to
Fc.gamma.RI. IgG2 residues at positions 233-236, substituted into
IgG1 and IgG4, reduced binding to Fc.gamma.RI by 10.sup.3-fold and
eliminated the human monocyte response to antibody-sensitized red
blood cells (Armour, K. L., et al., Eur. J. Immunol. 29 (1999)
2613-2624).
[0225] -Fc.gamma.RII (CD32) binds complexed IgG with medium to low
affinity and is widely expressed. This receptor can be divided into
two sub-types, Fc.gamma.RIIA and Fc.gamma.RIIB Fc.gamma.RIIA is
found on many cells involved in killing (e.g. macrophages,
monocytes, neutrophils) and seems able to activate the killing
process. Fc.gamma.RIIB seems to play a role in inhibitory processes
and is found on B cells, macrophages and on mast cells and
eosinophils. On B-cells it seems to function to suppress further
immunoglobulin production and isotype switching to, for example,
the IgE class. On macrophages, Fc.gamma.RIIB acts to inhibit
phagocytosis as mediated through Fc.gamma.RIIA. On eosinophils and
mast cells the B-form may help to suppress activation of these
cells through IgE binding to its separate receptor. Reduced binding
for Fc.gamma.RIIA is found e.g. for antibodies comprising an IgG
Fc-region with mutations at least at one of the amino acid residues
E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292,
and K414 (numbering according to EU index of Kabat).
[0226] Fc.gamma.RIII (CD16) binds IgG with medium to low affinity
and exists as two types. Fc.gamma.RIIIA is found on NK cells,
macrophages, eosinophils and some monocytes and T cells and
mediates ADCC. Fc.gamma.RIIIB is highly expressed on neutrophils.
Reduced binding to Fc.gamma.RIIIA is found e.g. for antibodies
comprising an IgG Fc-region with mutation at least at one of the
amino acid residues E233-G236, P238, D265, N297, A327, P329, D270,
Q295, A327, 5239, E269, E293, Y296, V303, A327, K338 and D376
(numbering according to EU index of Kabat).
[0227] Mapping of the binding sites on human IgG1 for Fc receptors,
the above mentioned mutation sites and methods for measuring
binding to Fc.gamma.RI and Fc.gamma.RIIA are described in Shields,
R. L., et al. J. Biol. Chem. 276 (2001) 6591-6604.
[0228] The term "ADCC" or "antibody-dependent cellular
cytotoxicity" is a function mediated by Fc receptor binding and
refers to lysis of target cells by an antibody as reported herein
in the presence of effector cells. The capacity of the antibody to
induce the initial steps mediating ADCC is investigated by
measuring their binding to Fey receptors expressing cells, such as
cells, recombinantly expressing Fc.gamma.RI and/or Fc.gamma.RIIA or
NK cells (expressing essentially Fc.gamma.RIIIA) In particular,
binding to Fc.gamma.R on NK cells is measured.
[0229] An "activating Fc receptor" is an Fc receptor that following
engagement by an Fc region of an antibody elicits signaling events
that stimulate the receptor-bearing cell to perform effector
functions. Activating Fc receptors include Fc.gamma.RIIIa (CD16a),
Fc.gamma.RI (CD64), Fc.gamma.RIIa (CD32), and Fc.alpha.RI (CD89). A
particular activating Fc receptor is human Fc.gamma.RIIIa (see
UniProt accession no. P08637, version 141).
[0230] The term "CD40", as used herein, refers to any native CD40
from any vertebrate source, including mammals such as primates
(e.g. humans) and rodents (e.g., mice and rats), unless otherwise
indicated. The term encompasses "full-length," unprocessed CD40 as
well as any form of CD40 that results from processing in the cell.
The term also encompasses naturally occurring variants of CD40,
e.g., splice variants or allelic variants. The amino acid sequence
of an exemplary human CD40 is shown in SEQ ID NO:1 (Uniprot P25942,
version 200) and the amino acid sequence of an exemplary mouse CD40
is shown in SEQ ID NO: 146 (Uniprot P27512, version 160). The CD40
antigen is a 50 kDa cell surface glycoprotein which belongs to the
Tumor Necrosis Factor Receptor (TNF-R) family. (Stamenkovic et al.
(1989), EMBO J. 8: 1403-10). CD40 is expressed in many normal and
tumor cell types, including B lymphocytes, dendritic cells,
monocytes, macrophages, thymus epithelium, endothelial cells,
fibroblasts, and smooth muscle cells. CD40 is expressed in all
B-lymphomas and in 70% of all solid tumors and is up-regulated in
antigen presenting cells (APCs) by maturation signals, such as
IFN-gamma and GM-CSF. CD40 activation also induces differentiation
of monocytes into functional dendritic cells (DCs) and enhances
cytolytic activity of NK cells through APC-CD40 induced cytokines.
Thus CD40 plays an essential role in the initiation and enhancement
of immune responses by inducing maturation of APCs, secretion of
helper cytokines, upregulation of costimulatory molecules, and
enhancement of effector functions.
[0231] The term "CD40 agonist" as used herein includes any moiety
that agonizes the CD40/CD40L interaction. CD40 as used in this
context refers preferably to human CD40, thus the CD40 agonist is
preferably an agonist of human CD40. Typically, the moiety will be
an agonistic CD40 antibody or antibody fragment.
[0232] The terms "anti-CD40 antibody", "anti-CD40", "CD40 antibody
and "an antibody that specifically binds to CD40" refer to an
antibody that is capable of binding CD40 with sufficient affinity
such that the antibody is useful as a diagnostic and/or therapeutic
agent in targeting CD40. In one aspect, the extent of binding of an
anti-CD40 antibody to an unrelated, non-CD40 protein is less than
about 10% of the binding of the antibody to CD40 as measured, e.g.,
by a radioimmunoassay (RIA) or flow cytometry (FACS). In certain
embodiments, an antibody that binds to CD40 has a dissociation
constant (K.sub.D) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10
nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001
nM (e.g. 10.sup.-6M or less, e.g. from 10.sup.-68M to 10.sup.13M,
e.g., from 10.sup.-8M to 10.sup.-10 M).
[0233] The term "peptide linker" refers to a peptide comprising one
or more amino acids, typically about 2 to 20 amino acids. Peptide
linkers are known in the art or are described herein. Suitable,
non-immunogenic linker peptides are, for example, (G.sub.4S).sub.n,
(SG.sub.4).sub.n or G.sub.4(SG.sub.4).sub.n peptide linkers,
wherein "n" is generally a number between 1 and 10, typically
between 2 and 4, in particular 2, i.e. the peptides selected from
the group consisting of GGGGS (SEQ ID NO: 147) GGGGSGGGGS (SEQ ID
NO:148), SGGGGSGGGG (SEQ ID NO:149) and GGGGSGGGGSGGGG (SEQ ID
NO:150), but also include the sequences GSPGSSSSGS (SEQ ID NO:151),
(G4S)3 (SEQ ID NO:152), (G4S).sub.4 (SEQ ID NO:153), GSGSGSGS (SEQ
ID NO:154), GSGSGNGS (SEQ ID NO:155), GGSGSGSG (SEQ ID NO:156),
GGSGSG (SEQ ID NO:157), GGSG (SEQ ID NO:158), GGSGNGSG (SEQ ID
NO:159), GGNGSGSG (SEQ ID NO:160) and GGNGSG (SEQ ID NO:161).
Peptide linkers of particular interest are (G4S) (SEQ ID NO:147),
(G.sub.4S).sub.2 or GGGGSGGGGS (SEQ ID NO:148), (G4S).sub.3 (SEQ ID
NO:152) and (G4S).sub.4 (SEQ ID NO:153).
[0234] The term "amino acid" as used within this application
denotes the group of naturally occurring carboxy a-amino acids
comprising alanine (three letter code: ala, one letter code: A),
arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D),
cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E),
glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine
(leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe,
F), proline (pro, P), serine (ser, S), threonine (thr, T),
tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
[0235] By "fused" or "connected" is meant that the components (e.g.
a heavy chain of an antibody and a Fab fragment) are linked by
peptide bonds, either directly or via one or more peptide
linkers.
[0236] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide (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 reference polypeptide
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. SAWI or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for aligning sequences, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are generated using the
sequence comparison computer program ALIGN-2. The ALIGN-2 sequence
comparison computer program was authored by Genentech, Inc., and
the source code has been filed with user documentation in the U.S.
Copyright Office, Washington D.C., 20559, where it is registered
under U.S. Copyright Registration No. TXU510087. The ALIGN-2
program is publicly available from Genentech, Inc., South San
Francisco, Calif., or may be compiled from the source code. The
ALIGN-2 program should be compiled for use on a UNIX operating
system, including digital UNIX V4.0D. All sequence comparison
parameters are set by the ALIGN-2 program and do not vary. In
situations where ALIGN-2 is employed for amino acid sequence
comparisons, the % amino acid sequence identity of a given amino
acid sequence A to, with, or against a given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence
A that has or comprises a certain % amino acid sequence identity
to, with, or against a given amino acid sequence B) is calculated
as follows:
100 times the fraction X/Y
[0237] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program ALIGN-2 in that
program's alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be appreciated that where the
length of amino acid sequence A is not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will
not equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0238] In certain embodiments, amino acid sequence variants of the
bispecific antigen binding molecules provided herein are
contemplated. For example, it may be desirable to improve the
binding affinity and/or other biological properties of the TNF
ligand trimer-containing antigen binding molecules. Amino acid
sequence variants of the TNF ligand trimer-containing antigen
binding molecules may be prepared by introducing appropriate
modifications into the nucleotide sequence encoding the molecules,
or by peptide synthesis. Such modifications include, for example,
deletions from, and/or insertions into and/or substitutions of
residues within the amino acid sequences of the antibody. Any
combination of deletion, insertion, and substitution can be made to
arrive at the final construct, provided that the final construct
possesses the desired characteristics, e.g., antigen-binding. Sites
of interest for substitutional mutagenesis include the HVRs and
Framework (FRs). Conservative substitutions are provided in Table B
under the heading "Preferred Substitutions" and further described
below in reference to amino acid side chain classes (1) to (6).
Amino acid substitutions may be introduced into the molecule of
interest and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
TABLE-US-00002 TABLE B Original Preferred Residue Exemplary
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0239] Amino acids may be grouped according to common side-chain
properties:
[0240] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0241] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0242] (3) acidic: Asp, Glu;
[0243] (4) basic: His, Lys, Arg;
[0244] (5) residues that influence chain orientation: Gly, Pro;
[0245] (6) aromatic: Trp, Tyr, Phe.
[0246] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0247] The term "amino acid sequence variants" includes substantial
variants wherein there are amino acid substitutions in one or more
hypervariable region residues of a parent antigen binding molecule
(e.g. a humanized or human antibody). Generally, the resulting
variant(s) selected for further study will have modifications
(e.g., improvements) in certain biological properties (e.g.,
increased affinity, reduced immunogenicity) relative to the parent
antigen binding molecule and/or will have substantially retained
certain biological properties of the parent antigen binding
molecule. An exemplary substitutional variant is an affinity
matured antibody, which may be conveniently generated, e.g., using
phage display-based affinity maturation techniques such as those
described herein. Briefly, one or more HVR residues are mutated and
the variant antigen binding molecules displayed on phage and
screened for a particular biological activity (e.g. binding
affinity). In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antigen
binding molecule to bind antigen. For example, conservative
alterations (e.g., conservative substitutions as provided herein)
that do not substantially reduce binding affinity may be made in
HVRs. A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as Arg, Asp, His, Lys,
and Glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antigen binding molecule complex to identify contact points
between the antibody and antigen. Such contact residues and
neighboring residues may be targeted or eliminated as candidates
for substitution. Variants may be screened to determine whether
they contain the desired properties.
[0248] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include bispecific antigen binding
molecules of the invention with an N-terminal methionyl residue.
Other insertional variants of the molecule include the fusion to
the N- or C-terminus to a polypeptide which increases the serum
half-life of the bispecific antigen binding molecules.
[0249] In certain embodiments, the bispecific antigen binding
molecules provided herein are altered to increase or decrease the
extent to which the antibody is glycosylated. Glycosylation
variants of the molecules may be conveniently obtained by altering
the amino acid sequence such that one or more glycosylation sites
is created or removed. Where the TNF ligand trimer-containing
antigen binding molecule comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in TNF
family ligand trimer-containing antigen binding molecule may be
made in order to create variants with certain improved properties.
In one aspect, variants of bispecific antigen binding molecules or
antibodies of the invention are provided having a carbohydrate
structure that lacks fucose attached (directly or indirectly) to an
Fc region. Such fucosylation variants may have improved ADCC
function, see e.g. US Patent Publication Nos. US 2003/0157108
(Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). In
another aspect, variants of the bispecific antigen binding
molecules or antibodies of the invention are provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region is bisected by GlcNAc. Such variants may
have reduced fucosylation and/or improved ADCC function., see for
example WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No.
6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Variants with at least one galactose residue in the oligosaccharide
attached to the Fc region are also provided. Such antibody variants
may have improved CDC function and are described, e.g., in WO
1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO
1999/22764 (Raju, S.).
[0250] In certain aspects, it may be desirable to create cysteine
engineered variants of the bispecific antigen binding molecules of
the invention, e.g., "thioMAbs," in which one or more residues of
the molecule are substituted with cysteine residues. In particular
aspects, the substituted residues occur at accessible sites of the
molecule. By substituting those residues with cysteine, reactive
thiol groups are thereby positioned at accessible sites of the
antibody and may be used to conjugate the antibody to other
moieties, such as drug moieties or linker-drug moieties, to create
an immunoconjugate. In certain aspects, any one or more of the
following residues may be substituted with cysteine: V205 (Kabat
numbering) of the light chain; A118 (EU numbering) of the heavy
chain; and S400 (EU numbering) of the heavy chain Fc region.
Cysteine engineered antigen binding molecules may be generated as
described, e.g., in U.S. Pat. No. 7,521,541.
[0251] The term "polynucleotide" refers to an isolated nucleic acid
molecule or construct, e.g. messenger RNA (mRNA), virally-derived
RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a
conventional phosphodiester bond or a non-conventional bond (e.g.
an amide bond, such as found in peptide nucleic acids (PNA). The
term "nucleic acid molecule" refers to any one or more nucleic acid
segments, e.g. DNA or RNA fragments, present in a
polynucleotide.
[0252] By "isolated" nucleic acid molecule or polynucleotide is
intended a nucleic acid molecule, DNA or RNA, which has been
removed from its native environment. For example, a recombinant
polynucleotide encoding a polypeptide contained in a vector is
considered isolated for the purposes of the present invention.
Further examples of an isolated polynucleotide include recombinant
polynucleotides maintained in heterologous host cells or purified
(partially or substantially) polynucleotides in solution. An
isolated polynucleotide includes a polynucleotide molecule
contained in cells that ordinarily contain the polynucleotide
molecule, but the polynucleotide molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location. Isolated RNA molecules
include in vivo or in vitro RNA transcripts of the present
invention, as well as positive and negative strand forms, and
double-stranded forms. Isolated polynucleotides or nucleic acids
according to the present invention further include such molecules
produced synthetically. In addition, a polynucleotide or a nucleic
acid may be or may include a regulatory element such as a promoter,
ribosome binding site, or a transcription terminator.
[0253] By a nucleic acid or polynucleotide having a nucleotide
sequence at least, for example, 95% "identical" to a reference
nucleotide sequence of the present invention, it is intended that
the nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence. These alterations of the reference sequence may
occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence. As a practical matter, whether any particular
polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to a nucleotide sequence of the present
invention can be determined conventionally using known computer
programs, such as the ones discussed above for polypeptides (e.g.
ALIGN-2).
[0254] The term "expression cassette" refers to a polynucleotide
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a target cell. The recombinant
expression cassette can be incorporated into a plasmid, chromosome,
mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
Typically, the recombinant expression cassette portion of an
expression vector includes, among other sequences, a nucleic acid
sequence to be transcribed and a promoter. In certain embodiments,
the expression cassette of the invention comprises polynucleotide
sequences that encode bispecific antigen binding molecules of the
invention or fragments thereof.
[0255] The term "vector" or "expression vector" is synonymous with
"expression construct" and refers to a DNA molecule that is used to
introduce and direct the expression of a specific gene to which it
is operably associated in a target cell. The term includes the
vector as a self-replicating nucleic acid structure as well as the
vector incorporated into the genome of a host cell into which it
has been introduced. The expression vector of the present invention
comprises an expression cassette. Expression vectors allow
transcription of large amounts of stable mRNA. Once the expression
vector is inside the target cell, the ribonucleic acid molecule or
protein that is encoded by the gene is produced by the cellular
transcription and/or translation machinery. In one embodiment, the
expression vector of the invention comprises an expression cassette
that comprises polynucleotide sequences that encode bispecific
antigen binding molecules of the invention or fragments
thereof.
[0256] The terms "host cell", "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein. A host cell is any
type of cellular system that can be used to generate the bispecific
antigen binding molecules of the present invention. Host cells
include cultured cells, e.g. mammalian cultured cells, such as CHO
cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63
mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells,
yeast cells, insect cells, and plant cells, to name only a few, but
also cells comprised within a transgenic animal, transgenic plant
or cultured plant or animal tissue.
[0257] An "effective amount" of an agent refers to the amount that
is necessary to result in a physiological change in the cell or
tissue to which it is administered.
[0258] A "therapeutically effective amount" of an agent, e.g. a
pharmaceutical composition, refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result. A therapeutically effective
amount of an agent for example eliminates, decreases, delays,
minimizes or prevents adverse effects of a disease.
[0259] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g. cows, sheep,
cats, dogs, and horses), primates (e.g. humans and non-human
primates such as monkeys), rabbits, and rodents (e.g. mice and
rats). Particularly, the individual or subject is a human.
[0260] The term "pharmaceutical composition" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0261] A "pharmaceutically acceptable excipient" refers to an
ingredient in a pharmaceutical composition, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable excipient includes, but is not limited to, a buffer, a
stabilizer, or a preservative.
[0262] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0263] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, the molecules
of the invention are used to delay development of a disease or to
slow the progression of a disease.
[0264] The term "cancer" as used herein refers to proliferative
diseases, such as lymphomas, lymphocytic leukemias, lung cancer,
non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung
cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the
head or neck, cutaneous or intraocular melanoma, uterine cancer,
ovarian cancer, rectal cancer, cancer of the anal region, stomach
cancer, gastric cancer, colon cancer, breast cancer, uterine
cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, cancer of the
bladder, cancer of the kidney or ureter, renal cell carcinoma,
carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer,
biliary cancer, neoplasms of the central nervous system (CNS),
spinal axis tumors, brain stem glioma, glioblastoma multiforme,
astrocytomas, schwanomas, ependymonas, medulloblastomas,
meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings
sarcoma, including refractory versions of any of the above cancers,
or a combination of one or more of the above cancers.
[0265] The term "chemotherapeutic agent" as used herein refers to a
chemical compound useful in the treatment of cancer. In one aspect,
the chemotherapeutic agent is an antimetabolite. In one aspect, the
antimetabolite is selected from the group consisting of
Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Cladribine,
Clofarabine, Fludarabine, Mercaptopurine, Pentostatin, Thioguanine,
Capecitabine, Cytarabine, Fluorouracil, Floxuridine, and
Gemcitabine. In one particular aspect, the antimetabolite is
capecitabine or gemcitabine. In another aspect, the antimetabolite
is fluorouracil. In one aspect, the chemotherapeutic agent is an
agent that affects microtubule formation. In one aspect, the agent
that affects microtubule formation is selected from the group
consisting of: paclitaxel, docetaxel, vincristine, vinblastine,
vindesine, vinorelbin, taxotere, etoposide, and teniposide. In
another aspect, the chemotherapeutic agent is an alkylating agent
such as cyclophosphamide. In one aspect, the chemotherapeutic agent
is a cytotoxic antibiotic such as a topoisomerase II inhibitor. In
one aspect, the topoisomerase II inhibitor is doxorubicin.
[0266] Bispecific Antibodies of the Invention
[0267] The invention provides novel bispecific antigen binding
molecules with particularly advantageous properties such as
producibility, stability, binding affinity, biological activity,
targeting efficiency, reduced toxicity, an extended dosage range
that can be given to a patient and thereby a possibly enhanced
efficacy.
[0268] Exemplary Bispecific Antigen Binding Molecules
[0269] In one aspect, the invention provides bispecific antigen
binding molecules, comprising [0270] (a) at least one antigen
binding domain capable of specific binding to CD40, and [0271] (b)
at least one antigen binding domain capable of specific binding to
a target cell antigen, and [0272] (c) a Fc region composed of a
first and a second subunit capable of stable association.
[0273] In a particular aspect, these bispecific antigen binding
molecules are characterized by targeted agonistic binding to CD40.
In particular, the bispecific antigen binding molecule is a CD40
agonist that is targeted against a tumor associated target cell
antigen. In another particular aspect, the bispecific antigen
binding molecules of the invention comprise a Fc region composed of
a first and a second subunit capable of stable association which
comprises mutations that reduce effector function. The use of a Fc
region comprising mutations that reduce or abolish effector
function will prevent unspecific agonism by crosslinking via Fc
receptors and will prevent ADCC of CD40.sup.+ cells.
[0274] The bispecific antigen binding molecules as described herein
possess the advantage over conventional antibodies capable of
specific binding to CD40 in that they selectively induce immune
response at the target cells, which are typically cancer cells or
tumor stroma. In one aspect, the tumor-associated target cell
antigen is selected from the group consisting of Fibroblast
Activation Protein (FAP), Melanoma-associated Chondroitin Sulfate
Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR),
Carcinoembryonic Antigen (CEA), CD19, CD20 and CD33.
[0275] In a particular aspect, the tumor-associated target cell
antigen is FAP.
[0276] These bispecific antigen binding molecules are characterized
by FAP-targeted agonistic binding to CD40. In the presence of
FAP-expressing cells the bispecific antigen binding molecules are
able to activate antigen presenting cells (APCs, Example 2.1), to
activate human B cells (Examples 2.1.1 and 2.1.3), human Daudi
cells (Example 2.1.2) and human monocyte-derived dendritic cells
(moDCs, Example 2.1.4)
[0277] In one aspect, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising (i) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:19, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:20, and (iii) CDR-H3 comprising the amino
acid sequence of SEQ ID NO:21, and a light chain variable region
(V.sub.LCD40) comprising (iv) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:22, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:23, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:24.
[0278] In another aspect, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising (i) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:27, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:28, and (iii) CDR-H3 comprising the amino
acid sequence of SEQ ID NO:29, and a light chain variable region
(V.sub.LCD40) comprising (iv) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:30, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:31, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:32.
[0279] In one aspect, the bispecific antigen binding molecule
comprises an antigen binding domain capable of specific binding to
CD40 and comprises a comprises a heavy chain variable region
(V.sub.HCD40) comprising the amino acid sequence of SEQ ID NO:25
and a light chain variable region (V.sub.LCD40) comprising the
amino acid sequence of SEQ ID NO:26.
[0280] In another aspect, the bispecific antigen binding molecule
comprises an antigen binding domain capable of specific binding to
CD40 and comprises a comprises a heavy chain variable region
(V.sub.HCD40) comprising the amino acid sequence of SEQ ID NO:33
and a light chain variable region (V.sub.LCD40) comprising the
amino acid sequence of SEQ ID NO:34.
[0281] In another aspect, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 comprises
[0282] (i) a heavy chain variable region (V.sub.HCD40) comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ
ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54
and SEQ ID NO:55, and
[0283] (ii) a light chain variable region (V.sub.LCD40) comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,
SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64.
[0284] In one aspect, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising the amino acid sequence of SEQ ID NO:47
and a light chain variable region (V.sub.LCD40) comprising the
amino acid sequence of SEQ ID NO:57.
[0285] In a further aspect, provided is a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to CD40 comprises
[0286] (i) a heavy chain variable region (V.sub.HCD40) comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:171, SEQ ID NO:172, SEQ ID NO:173 and SEQ ID NO:174, and
[0287] (ii) a light chain variable region (V.sub.LCD40) comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178.
[0288] In one aspect, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 comprises
[0289] (a) a VH comprising the amino acid sequence of SEQ ID NO:171
and a VL comprising the amino acid sequence of SEQ ID NO:175,
or
[0290] (b) a VH comprising the amino acid sequence of SEQ ID NO:173
and a VL comprising the amino acid sequence of SEQ ID NO:177,
or
[0291] (c) a VH comprising the amino acid sequence of SEQ ID NO:174
and a VL comprising the amino acid sequence of SEQ ID NO:178,
or
[0292] (d) a VH comprising the amino acid sequence of SEQ ID NO:171
and a VL comprising the amino acid sequence of SEQ ID NO:177,
or
[0293] (e) a VH comprising the amino acid sequence of SEQ ID NO:171
and a VL comprising the amino acid sequence of SEQ ID NO:178,
or
[0294] (f) a VH comprising the amino acid sequence of SEQ ID NO:173
and a VL comprising the amino acid sequence of SEQ ID NO:175,
or
[0295] (g) a VH comprising the amino acid sequence of SEQ ID NO:173
and a VL comprising the amino acid sequence of SEQ ID NO:178,
or
[0296] (h) a VH comprising the amino acid sequence of SEQ ID NO:174
and a VL comprising the amino acid sequence of SEQ ID NO:175,
or
[0297] (i) a VH comprising the amino acid sequence of SEQ ID NO:174
and a VL comprising the amino acid sequence of SEQ ID NO:177,
or
[0298] (j) a VH comprising the amino acid sequence of SEQ ID NO:171
and a VL comprising the amino acid sequence of SEQ ID NO:176,
or
[0299] (k) a VH comprising the amino acid sequence of SEQ ID NO:172
and a VL comprising the amino acid sequence of SEQ ID NO:175,
or
[0300] (l) a VH comprising the amino acid sequence of SEQ ID NO:172
and a VL comprising the amino acid sequence of SEQ ID NO:176,
or
[0301] (m) a VH comprising the amino acid sequence of SEQ ID NO:172
and a VL comprising the amino acid sequence of SEQ ID NO:177,
or
[0302] (n) a VH comprising the amino acid sequence of SEQ ID NO:172
and a VL comprising the amino acid sequence of SEQ ID NO:178,
or
[0303] (o) a VH comprising the amino acid sequence of SEQ ID NO:173
and a VL comprising the amino acid sequence of SEQ ID NO:176,
or
[0304] (p) a VH comprising the amino acid sequence of SEQ ID NO:174
and a VL comprising the amino acid sequence of SEQ ID NO:176.
[0305] In a particular aspect, provided is a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to CD40 comprises a VH comprising the amino acid
sequence of SEQ ID NO:171 and a VL comprising the amino acid
sequence of SEQ ID NO:175.
[0306] In yet another aspect, provided is a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to CD40 comprises
[0307] (i) a heavy chain variable region (V.sub.HCD40) comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183
and SEQ ID NO:184, and
[0308] (ii) a light chain variable region (V.sub.LCD40) comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:185, SEQ ID NO:186, SEQ ID NO:187, and SEQ ID NO:188.
[0309] In one aspect, provided is a bispecific antigen binding
molecule, wherein the antigen binding domain capable of specific
binding to CD40 comprises
[0310] (a) a VH comprising the amino acid sequence of SEQ ID NO:179
and a VL comprising the amino acid sequence of SEQ ID NO:185,
or
[0311] (b) a VH comprising the amino acid sequence of SEQ ID NO:180
and a VL comprising the amino acid sequence of SEQ ID NO:185,
or
[0312] (c) a VH comprising the amino acid sequence of SEQ ID NO:181
and a VL comprising the amino acid sequence of SEQ ID NO:185,
or
[0313] (d) a VH comprising the amino acid sequence of SEQ ID NO:182
and a VL comprising the amino acid sequence of SEQ ID NO:185,
or
[0314] (e) a VH comprising the amino acid sequence of SEQ ID NO:179
and a VL comprising the amino acid sequence of SEQ ID NO:186,
or
[0315] (f) a VH comprising the amino acid sequence of SEQ ID NO:180
and a VL comprising the amino acid sequence of SEQ ID NO:186,
or
[0316] (g) a VH comprising the amino acid sequence of SEQ ID NO:181
and a VL comprising the amino acid sequence of SEQ ID NO:186,
or
[0317] (h) a VH comprising the amino acid sequence of SEQ ID NO:182
and a VL comprising the amino acid sequence of SEQ ID NO:186,
or
[0318] (i) a VH comprising the amino acid sequence of SEQ ID NO:183
and a VL comprising the amino acid sequence of SEQ ID NO:187,
or
[0319] (j) a VH comprising the amino acid sequence of SEQ ID NO:183
and a VL comprising the amino acid sequence of SEQ ID NO:188,
or
[0320] (k) a VH comprising the amino acid sequence of SEQ ID NO:184
and a VL comprising the amino acid sequence of SEQ ID NO:187,
or
[0321] (l) a VH comprising the amino acid sequence of SEQ ID NO:184
and a VL comprising the amino acid sequence of SEQ ID NO:188.
[0322] In a particular aspect, provided is a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to CD40 comprises a VH comprising the amino acid
sequence of SEQ ID NO:179 and a VL comprising the amino acid
sequence of SEQ ID NO:185 or wherein the antigen binding domain
capable of specific binding to CD40 comprises a VH comprising the
amino acid sequence of SEQ ID NO:182 and a VL comprising the amino
acid sequence of SEQ ID NO:185.
[0323] Bispecific Antigen Binding Molecules wherein the Target Cell
Antigen is FAP
[0324] In a particular aspect, the target cell antigen is
Fibroblast Activation Protein (FAP). FAP binding moieties have been
described in WO 2012/02006 which is included by reference in its
entirety. FAP binding moieties of particular interest are described
below.
[0325] In one aspect, the invention provides a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to FAP binds to a polypeptide comprising, or
consisting of, the amino acid sequence of SEQ ID NO:2.
[0326] In another aspect, the invention provides a bispecific
antigen binding molecule, wherein the antigen binding domain
capable of specific binding to Fibroblast Activation Protein (FAP)
comprises [0327] (a) a heavy chain variable region (V.sub.HFAP)
comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID
NO:3, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:4, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:5, and a light chain variable region (V.sub.LFAP) comprising
(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:6, (v)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:7, and (vi)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, or [0328]
(b) a heavy chain variable region (V.sub.HFAP) comprising (i)
CDR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii)
CDR-H2 comprising the amino acid sequence of SEQ ID NO:12, and
(iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:13,
and a a light chain variable region (V.sub.LFAP) comprising (iv)
CDR-L1 comprising the amino acid sequence of SEQ ID NO:14, (v)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:15, and (vi)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:16.
[0329] In particular, provided is a bispecific antigen binding
molecule, wherein the heavy chain variable region (V.sub.HFAP)
comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID
NO:3, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:4, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:5, and the light chain variable region (V.sub.LFAP) comprising
(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:6, (v)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:7, and (vi)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:8. In
another aspect, the antigen binding domain capable of specific
binding to FAP comprises a heavy chain variable region (V.sub.HFAP)
comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID
NO:11, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:12, and (iii) CDR-H3 comprising the amino acid sequence of
SEQ
[0330] ID NO:13, and a a light chain variable region (V.sub.LFAP)
comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:14, (v) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:15, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:16.
[0331] In a further aspect, provided is a bispecific antigen
binding molecule, wherein the antigen binding domain capable of
specific binding to FAP comprises (a) a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:9, and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:10, or (b) a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:17, and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:18.
[0332] In one aspect, the antigen binding domain capable of
specific binding to FAP comprises a heavy chain variable region VH
comprising the amino acid sequence of SEQ ID NO: 9 and a light
chain variable region comprising an amino acid sequence of SEQ ID
NO: 10 or the antigen binding domain capable of specific binding to
FAP comprises a heavy chain variable region VH comprising an amino
acid sequence of SEQ ID NO:17 and a light chain variable region
comprising an amino acid sequence of SEQ ID NO:18.
[0333] Bispecific Antigen Binding Molecules Binding to CD40 and
FAP
[0334] In another aspect, provided is a bispecific antigen binding
molecule, comprising
[0335] (i) at least one antigen binding domain capable of specific
binding to CD40, comprising a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:25, SEQ ID NO:45, SEQ ID NO:46, SEQ
ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, and a
light chain variable region (V.sub.LCD40) comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:26, SEQ ID
NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ
ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, and
[0336] (ii) at least one antigen binding domain capable of specific
binding to FAP, comprising a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:9 and a
light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:10, or a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:17 and
a light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:18.
[0337] In a further aspect, provided is a bispecific antigen
binding molecule, comprising
[0338] (i) at least one antigen binding domain capable of specific
binding to CD40, comprising a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173,
SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID
NO:182, SEQ ID NO:183 and SEQ ID NO:184, and a light chain variable
region (V.sub.LCD40) comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID
NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187
and SEQ ID NO:188, and
[0339] (ii) at least one antigen binding domain capable of specific
binding to FAP, comprising a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:9 and a
light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:10, or a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:17 and
a light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:18.
[0340] In a particular aspect, provided is a bispecific antigen
binding molecule, comprising
[0341] (i) at least one antigen binding domain capable of specific
binding to CD40, comprising a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence of SEQ ID NO:171
and a light chain variable region (V.sub.LCD40) comprising an amino
acid sequence of SEQ ID NO:175, and
[0342] (ii) at least one antigen binding domain capable of specific
binding to FAP, comprising a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:9 and a
light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:10, or a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:17 and
a light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:18.
[0343] In another particular aspect, provided is a bispecific
antigen binding molecule, comprising
[0344] (i) at least one antigen binding domain capable of specific
binding to CD40, comprising a heavy chain variable region
(V.sub.HCD40) comprising an amino acid of SEQ ID NO:179 or SEQ
ID
[0345] NO:182 and a light chain variable region (V.sub.LCD40)
comprising an amino acid sequence of SEQ ID NO:185, and
[0346] (ii) at least one antigen binding domain capable of specific
binding to FAP, comprising a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:9 and a
light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:10, or a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:17 and
a light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:18.
[0347] In a further aspect, provided is a bispecific antigen
binding molecule, wherein [0348] (i) the antigen binding domain
capable of specific binding to CD40 comprises a heavy chain
variable region (V.sub.HCD40) comprising the amino acid sequence of
SEQ ID NO: 25 and a light chain variable region (V.sub.LCD40)
comprising an amino acid sequence of SEQ ID NO: 26 and [0349] (ii)
the antigen binding domain capable of specific binding to FAP
comprises a heavy chain variable region VH comprising an amino acid
sequence of SEQ ID NO:9 and a light chain variable region
comprising an amino acid sequence of SEQ ID NO:10.
[0350] Furthermore, provided is a bispecific antigen binding
molecule, wherein [0351] (i) the antigen binding domain capable of
specific binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising the amino acid sequence of SEQ ID NO: 25
and a light chain variable region (V.sub.LCD40) comprising an amino
acid sequence of SEQ ID NO: 26 and [0352] (ii) the antigen binding
domain capable of specific binding to FAP comprises a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:17 and a light chain variable region comprising an amino acid
sequence of SEQ ID NO:18.
[0353] In another aspect, provided is a bispecific antigen binding
molecule, wherein [0354] (i) the antigen binding domain capable of
specific binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising the amino acid sequence of SEQ ID NO: 47
and a light chain variable region (V.sub.LCD40) comprising an amino
acid sequence of SEQ ID NO: 57 and [0355] (ii) the antigen binding
domain capable of specific binding to FAP comprises a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:17 and a light chain variable region comprising an amino acid
sequence of SEQ ID NO:18.
[0356] In another aspect, provided is a bispecific antigen binding
molecule, wherein [0357] (i) the antigen binding domain capable of
specific binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173,
SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID
NO:182, SEQ ID NO:183 and SEQ ID NO:184, and a light chain variable
region (V.sub.LCD40) comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID
NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187
and SEQ ID NO:188, and [0358] (ii) the antigen binding domain
capable of specific binding to FAP comprises a heavy chain variable
region VH comprising an amino acid sequence of SEQ ID NO:17 and a
light chain variable region comprising an amino acid sequence of
SEQ ID NO:18.
[0359] Bispecific, Monovalent Antigen Binding Molecules (1+1
format)
[0360] In one aspect, the invention relates to bispecifc antigen
binding molecules comprising (a) one antigen binding domain capable
of specific binding to a CD40, (b) one antigen binding domain
capable of specific binding to a target cell antigen, and (c) a Fc
domain composed of a first and a second subunit capable of stable
association.
[0361] In a particular aspect, provided is a bispecific antigen
binding molecule, wherein said molecule comprises (a) a first Fab
fragment capable of specific binding to CD40, (b) a second Fab
fragment capable of specific binding to a target cell antigen, and
(c) a Fc domain composed of a first and a second subunit capable of
stable association. In one aspect, the target cell antigen is
FAP.
[0362] In one aspect, provided is a bispecific antigen binding
molecule, wherein said molecule comprises [0363] (i) a first Fab
fragment capable of specific binding to CD40, comprising a heavy
chain variable region (V.sub.HCD40) comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:25, SEQ ID
NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ
ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54
and SEQ ID NO:55, and a light chain variable region (V.sub.LCD40)
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:26, SEQ ID NO:56, SEQ ID NO:57, SEQ ID
NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ
ID NO:63 and SEQ ID NO:64, and [0364] (ii) a second Fab fragment
capable of specific binding to FAP, comprising a heavy chain
variable region (V.sub.HFAP) comprising an amino acid sequence of
SEQ ID NO:9 and a light chain variable region (V.sub.LFAP)
comprising an amino acid sequence of SEQ ID NO:10, or a heavy chain
variable region (V.sub.HFAP) comprising an amino acid sequence of
SEQ ID NO:17 and a light chain variable region (V.sub.LFAP)
comprising an amino acid sequence of SEQ ID NO:18.
[0365] In one aspect, provided is a bispecific antigen binding
molecule comprising a first heavy chain (HC1) comprising the amino
acid sequence of SEQ ID NO:141, a second heavy chain (HC2)
comprising the amino acid sequence of SEQ ID NO:140, a first light
chain comprising the amino acid sequence of SEQ ID NO:138 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:137.
[0366] In another aspect, provided is a bispecific antigen binding
molecule, wherein said molecule comprises [0367] (i) a first Fab
fragment capable of specific binding to CD40, comprising a heavy
chain variable region (V.sub.HCD40) comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:25, SEQ ID
NO:33, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174,
SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID
NO:183 and SEQ ID NO:184, and a light chain variable region
(V.sub.LCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:175, SEQ
ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID
NO:186, SEQ ID NO:187 and SEQ ID NO:188, and [0368] (ii) a second
Fab fragment capable of specific binding to FAP, comprising a heavy
chain variable region (V.sub.HFAP) comprising an amino acid
sequence of SEQ ID NO:9 and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence of SEQ ID NO:10, or
a heavy chain variable region (V.sub.HFAP) comprising an amino acid
sequence of SEQ ID NO:17 and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence of SEQ ID NO:18.
[0369] In one particular aspect, provided is a bispecific antigen
binding molecule, wherein said molecule comprises [0370] (i) a
first Fab fragment capable of specific binding to CD40, comprising
a heavy chain variable region (V.sub.HCD40) comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:171,
SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ ID
NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID
NO:184, and a light chain variable region (V.sub.LCD40) comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185,
SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:188, and [0371] (ii) a
second Fab fragment capable of specific binding to FAP, comprising
a heavy chain variable region (V.sub.HFAP) comprising an amino acid
sequence of SEQ ID NO:9 and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence of SEQ ID NO:10, or
a heavy chain variable region (V.sub.HFAP) comprising an amino acid
sequence of SEQ ID NO:17 and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence of SEQ ID NO:18.
[0372] In a particular aspect, provided is a bispecific antigen
binding molecule, wherein said molecule comprises (i) a first Fab
fragment capable of specific binding to CD40, comprising a heavy
chain variable region (V.sub.HCD40) comprising an amino acid
sequence of SEQ ID NO:171 and a light chain variable region
(V.sub.LCD40) comprising an amino acid sequence of SEQ ID NO:175,
and (ii) a second Fab fragment capable of specific binding to FAP,
comprising a heavy chain variable region (V.sub.HFAP) comprising an
amino acid sequence of SEQ ID NO:17 and a light chain variable
region (V.sub.LFAP) comprising an amino acid sequence of SEQ ID
NO:18.
[0373] In another particular aspect, provided is a bispecific
antigen binding molecule, wherein said molecule comprises (i) a
first Fab fragment capable of specific binding to CD40, comprising
a heavy chain variable region (V.sub.HCD40) comprising an amino
acid sequence of SEQ ID NO:179 or SEQ ID NO:182, and a light chain
variable region (V.sub.LCD40) comprising an amino acid sequence of
SEQ ID NO:185, and (ii) a second Fab fragment capable of specific
binding to FAP, comprising a heavy chain variable region
(V.sub.HFAP) comprising an amino acid sequence of SEQ ID NO:17 and
a light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:18.
[0374] In a particular aspect, provided is a bispecific antigen
binding molecule comprising a first heavy chain (HC1) comprising
the amino acid sequence of SEQ ID NO:163, a second heavy chain
(HC2) comprising the amino acid sequence of SEQ ID NO:164, a first
light chain comprising the amino acid sequence of SEQ ID NO:165 and
a second light chain comprising the amino acid sequence of SEQ ID
NO:162.
[0375] Bispecific Antigen Binding Molecules Bivalent for Binding to
CD40 and Monovalent for Binding to the Target Cell Antigen (2+1
Format)
[0376] In another aspect, the invention provides a bispecific
antigen binding molecule comprising [0377] (a) two antigen binding
domains capable of specific binding to CD40, [0378] (b) one antigen
binding domain capable of specific binding to the target cell
antigen, and [0379] (c) a Fc domain composed of a first and a
second subunit capable of stable association.
[0380] Thus, provided is a bispecific antigen binding molecule,
wherein the bispecific antigen binding molecule is bivalent for
CD40 and monovalent for the target cell antigen.
[0381] In one aspect, the bispecific antigen binding molecule
comprises [0382] (a) two light chains and two heavy chains of an
antibody comprising two Fab fragments capable of specific binding
to CD40 and the Fc domain, and [0383] (b) a VH and VL domain
capable of specific binding to a target cell antigen, wherein the
VH domain and the VL domain are each connected via a peptide linker
to one of the C-termini of the two heavy chains.
[0384] In a particular aspect, the peptide linker comprises an
amino acid sequence selected from SEQ ID NO:147, SEQ ID NO:148, SEQ
ID NO:152 and SEQ ID NO:153. More particularly, the peptide linker
comprises the SEQ ID NO:153.
[0385] In a particular aspect, the bispecific antigen binding
molecule comprises [0386] (a) two light chains and two heavy chains
of an antibody comprising two Fab fragments capable of specific
binding to CD40 and the Fc domain, and [0387] (b) a VH and VL
domain capable of specific binding to a target cell antigen,
wherein the VH domain is connected via a peptide linker to the
C-terminus of one of the heavy chains and wherein the VL domain is
connected via a peptide linker to the C-terminus of the second
heavy chain.
[0388] In another particular aspect, the bispecific antigen binding
molecule comprises [0389] (a) two light chains and two heavy chains
of an antibody comprising two Fab fragments capable of specific
binding to CD40 and the Fc domain, and [0390] (b) a VH and VL
domain capable of specific binding to a target cell antigen,
wherein the VH domain is connected via a peptide linker to the
C-terminus of the Fc knob heavy chain and wherein the VL domain is
connected via a peptide linker to the C-terminus of the Fc hole
heavy chain.
[0391] In one aspect, the bispecific antigen binding molecule
comprises [0392] (a) two light chains and two heavy chains of an
antibody comprising two Fab fragments capable of specific binding
to CD40 and the Fc domain, and [0393] (b) a VH and VL domain
capable of specific binding to a target cell antigen, wherein the
VL domain is connected via a peptide linker to the C-terminus of
the Fc knob heavy chain and wherein the VH domain is connected via
a peptide linker to the C-terminus of the Fc hole heavy chain.
[0394] In one aspect, the invention relates to a bispecific antigen
binding molecule, comprising [0395] (a) two Fab fragments capable
of specific binding to CD40 connected to a Fc region, and [0396]
(b) one antigen binding domain capable of specific binding to FAP
connected to the C-terminus of the Fc region.
[0397] In a particular aspect, the invention provides a bispecific
antigen binding molecule comprising [0398] (a) two light chains and
two heavy chains of an antibody comprising two Fab fragments
capable of specific binding to CD40, and a Fc region, and [0399]
(b) a VH and a VL of an antigen binding domain capable specific
binding to FAP, wherein the
[0400] VH is connected to the C-terminus of one of the two heavy
chains of (a), and wherein the VL is connected to the C-terminus of
the other of the two heavy chains of (a).
[0401] In another aspect, the invention relates to a bispecific
antigen binding molecule, comprising [0402] (a) two heavy chains,
each heavy chain comprising a VH and CH1 domain of a Fab fragment
capable of specific binding to CD40 and a Fc region subunit, [0403]
(b) two light chains, each light chain comprising a VL and CL
domain of a Fab fragment capable of specific binding to CD40, and
[0404] (c) a VH and a VL of an antigen binding domain capable of
specific binding to FAP, wherein the VH is connected to the
C-terminus of one of the two heavy chains of (a), and wherein the
VL is connected to the C-terminus of the other of the two heavy
chains of (a)
[0405] In particular, the VH domain is a heavy chain variable
region (V.sub.HFAP) comprising an amino acid sequence of SEQ ID
NO:9 or of SEQ ID NO:17 and the VL domain is a light chain variable
region (V.sub.LFAP) comprising an amino acid sequence of SEQ ID
NO:10 or of SEQ ID NO:18. More particularly, the VH domain is a
heavy chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:17 and the VL domain is a light chain
variable region (V.sub.LFAP) comprising an amino acid sequence of
SEQ ID NO:18.
[0406] In a particular aspect, the invention provides a bispecific
antigen binding molecule comprising [0407] (a) two light chains,
each comprising the amino acid sequence of SEQ ID NO:82, a first
heavy chain comprising the amino acid sequence of SEQ ID NO:88, and
a second heavy chain comprising the amino acid sequence of SEQ ID
NO:89, or [0408] (b) two light chains, each comprising the amino
acid sequence of SEQ ID NO:133, a first heavy chain comprising the
amino acid sequence of SEQ ID NO:134, and a second heavy chain
comprising the amino acid sequence of SEQ ID NO:135.
[0409] In another particular aspect, the bispecific antigen binding
molecule comprises [0410] (a) two light chains and two heavy chains
of an antibody comprising two Fab fragments capable of specific
binding to CD40 and the Fc domain, and [0411] (b) a Fab fragment
capable of specific binding to a target cell antigen, wherein the
Fab fragment is connected via a peptide linker to the C-terminus of
one of the heavy chains.
[0412] In one aspect, provided is a bispecific antigen binding
molecule, wherein the bispecific antigen binding molecule comprises
[0413] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40,
and a Fc region subunit, [0414] (b) two light chains, each light
chain comprising a VL and CL domain of a Fab fragment capable of
specific binding to CD40, and [0415] (c) one Fab fragment capable
of specific binding to FAP, wherein the Fab fragment is connected
to the C-terminus of one of the two heavy chains of (a).
[0416] In particular, the Fab fragment capable of specific binding
is a crossover fab fragment.
[0417] In one aspect, the invention provides a bispecific antigen
binding molecule comprising two light chains, each comprising the
amino acid sequence of SEQ ID NO:137, one light chain comprising
the amino acid sequence of SEQ ID NO:138, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:139, and a second
heavy chain comprising the amino acid sequence of SEQ ID
NO:136.
[0418] In a further aspect, provided is a bispecific antigen
binding molecule, comprising
[0419] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40
and a Fc region subunit,
[0420] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0421] (c) a crossover fab fragment capable of specific binding to
FAP comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL
chain is connected to the C-terminus of one of the two heavy chains
of (a).
[0422] In one aspect, the VH-CL chain is connected to the
C-terminus of the FC knob heavy chain.
[0423] In another aspect, provided is a bispecific antigen binding
molecule, comprising
[0424] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40
and a Fc region subunit,
[0425] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0426] (c) a crossover fab fragment capable of specific binding to
FAP comprising a VL-CH1 chain and a VH-CL chain, wherein the VL-CH1
chain is connected to the C-terminus of one of the two heavy chains
of (a).
[0427] In one aspect, the VL-CH1 chain is connected to the
C-terminus of the FC knob heavy chain.
[0428] In a particular aspect, the invention provides a bispecific
antigen binding molecule comprising [0429] (a) two light chains,
each comprising the amino acid sequence of SEQ ID NO:165, one light
chain comprising the amino acid sequence of SEQ ID NO:162, a first
heavy chain comprising the amino acid sequence of SEQ ID NO:167,
and a second heavy chain comprising the amino acid sequence of SEQ
ID NO:168, or [0430] (b) two light chains, each comprising the
amino acid sequence of SEQ ID NO:248, one light chain comprising
the amino acid sequence of SEQ ID NO:162, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:251, and a second
heavy chain comprising the amino acid sequence of SEQ ID NO:252, or
[0431] (c) two light chains, each comprising the amino acid
sequence of SEQ ID NO:248, one light chain comprising the amino
acid sequence of SEQ ID NO:138, a first heavy chain comprising the
amino acid sequence of SEQ ID NO:253, and a second heavy chain
comprising the amino acid sequence of SEQ ID NO:252, or [0432] (d)
two light chains, each comprising the amino acid sequence of SEQ ID
NO:248, one light chain comprising the amino acid sequence of SEQ
ID NO:254, a first heavy chain comprising the amino acid sequence
of SEQ ID NO:255, and a second heavy chain comprising the amino
acid sequence of SEQ ID NO:252, or [0433] (e) two light chains,
each comprising the amino acid sequence of SEQ ID NO:256, one light
chain comprising the amino acid sequence of SEQ ID NO:254, a first
heavy chain comprising the amino acid sequence of SEQ ID NO:257,
and a second heavy chain comprising the amino acid sequence of SEQ
ID NO:258.
[0434] Bispecific Antigen Binding Molecules in Head-to-Tail Format
(2+1)
[0435] In another aspect, provided is a bispecific antigen binding
molecule, comprising
[0436] (a) a heavy chain comprising a VH and CH1 domain of a Fab
fragment capable of specific binding to CD40 and a Fc region
subunit,
[0437] (b) a heavy chain comprising a VH and CH1 domain of a Fab
fragment capable of specific binding to CD40, a VL and CH1 domain
of a Fab fragment capable of specific binding to FAP and a Fc
region subunit,
[0438] (c) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0439] (d) a light chain comprising a VH and CL domain of a Fab
fragment capable of specific binding to FAP.
[0440] In particular, provided is a bispecific antigen binding
molecule comprising a first heavy chain comprising the amino acid
sequence of SEQ ID NO:164, a second heavy chain comprising the
amino acid sequence of SEQ ID NO:166, two light chains each
comprising the amino acid sequence of SEQ ID NO:165 and a light
chain comprising the amino acid sequence of SEQ ID NO:162.
[0441] Bispecific Antigen Binding Molecules Bivalent for Binding to
CD40 and Bivalent for Binding to the Target Cell Antigen (2+2
Format)
[0442] In another aspect, the invention provides a bispecific
antigen binding molecule comprising [0443] (a) two antigen binding
domains capable of specific binding to CD40, [0444] (b) two antigen
binding domains capable of specific binding to the target cell
antigen, and [0445] (c) a Fc domain composed of a first and a
second subunit capable of stable association.
[0446] Thus, provided is a bispecific antigen binding molecule,
wherein the bispecific antigen binding molecule is bivalent for
CD40 and bivalent for the target cell antigen.
[0447] In one aspect, provided is a bispecific antigen binding
molecule, wherein the bispecific antigen binding molecule
comprises
[0448] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40,
and a Fc region subunit,
[0449] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0450] (c) two Fab fragments capable of specific binding to FAP,
wherein one of the Fab fragments is connected to the C-terminus of
one of the two heavy chains of (a), and the other of the Fab
fragments is connected to the C-terminus of the other of the two
heavy chains of (a).
[0451] In a particular aspect, the invention provides a bispecific
antigen binding molecule comprising [0452] (a) two light chains,
each comprising the amino acid sequence of SEQ ID NO:86, two light
chains, each comprising the amino acid sequence of SEQ ID NO:87,
and two heavy chains, each comprising the amino acid sequence of
SEQ ID NO:90, or [0453] (b) two light chains, each comprising the
amino acid sequence of SEQ ID NO:137, two light chains, each
comprising the amino acid sequence of SEQ ID NO:138, and two heavy
chains, each comprising the amino acid sequence of SEQ ID
NO:136.
[0454] Bispecific Antigen Binding Molecules Tetravalent for Binding
to CD40 and Monovalent for Binding to the Target Cell Antigen (4+1
Format)
[0455] In another aspect, the invention provides a bispecific
antigen binding molecule comprising [0456] (a) four antigen binding
domains capable of specific binding to CD40, [0457] (b) one antigen
binding domain capable of specific binding to a target cell
antigen, and [0458] (c) a Fc domain composed of a first and a
second subunit capable of stable association:
[0459] Thus, provided is a bispecific antigen binding molecule,
wherein the bispecific antigen binding molecule is tetravalent for
CD40 and monovalent for the target cell antigen.
[0460] In one aspect, provided is a bispecific antigen binding
molecule, wherein the four antigen binding domains capable of
specific binding to CD40 are Fab fragments and each two thereof are
fused to each other at the heavy chain, optionally via a peptide
linker. In a particular aspect, the peptide linker comprises the
amino acid sequence of SEQ ID NO:148. More particularly, the
antigen binding molecule comprises two heavy chains comprising each
a VHCH1-peptide linker-VHCH1 fragment. In a particular aspect, the
peptide linker has the amino acid sequence of SEQ ID NO:148.
[0461] In another aspect, a bispecific antigen binding molecule is
provided, wherein the antigen binding domain capable of specific
binding to a target cell antigen comprises a VH and VL domain and
wherein the VH domain is connected via a peptide linker to the
C-terminus of the first subunit of the Fc domain and the VL domain
is connected via a peptide linker to the C-terminus of the second
subunit of the Fc domain.
[0462] In a particular aspect, the bispecific antigen binding
molecule comprises
[0463] (a) four light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to
CD40,
[0464] (b) two heavy chains, wherein each of the heavy chain
comprises a VH and CH1 domain of a Fab fragment capable of specific
binding to CD40 fused to a VH and CH1 domain of a second Fab
fragment capable of specific binding to CD40, and a Fc region
subunit, and
[0465] (c) a VH and VL domain capable of specific binding to a
target cell antigen, wherein the VH domain is connected via a
peptide linker to the C-terminus of one of the heavy chains and
wherein the VL domain is connected via a peptide linker to the
C-terminus of the second heavy chain.
[0466] In a particular aspect, the peptide linker comprises an
amino acid sequence selected from SEQ ID NO:147, SEQ ID NO:148, SEQ
ID NO:152 and SEQ ID NO:153. More particularly, the peptide linker
comprises the SEQ ID NO:153.
[0467] In one aspect, provided is a bispecific antigen binding
molecule comprising
[0468] (a) four light chains, each light chain comprising a light
chain variable region (V.sub.LCD40) comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:26, SEQ ID
NO:34, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178,
SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:188,
[0469] (b) two heavy chains, wherein each of the heavy chain
comprises VH-CH1-VH-CH1 and a Fc region subunit, and wherein both
VH domains comprise a heavy chain variable region (V.sub.HCD40)
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:171, SEQ ID
NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180,
SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184,
and
[0470] (c) a VH and VL domain capable of specific binding to a
target cell antigen, wherein the VH domain is connected via a
peptide linker to the C-terminus of one of the heavy chains and
wherein the VL domain is connected via a peptide linker to the
C-terminus of the second heavy chain.
[0471] In one aspect, the invention relates to a bispecific antigen
binding molecule, comprising [0472] (a) four Fab fragments capable
of specific binding to CD40, (b) a VH and a VL domain capable of
specific binding to FAP, and (c) a Fc domain composed of a first
and a second subunit capable of stable association.
[0473] In particular, the VH domain is a heavy chain variable
region (V.sub.HFAP) comprising an amino acid sequence of SEQ ID
NO:9 or of SEQ ID NO:17 and the VL domain is a light chain variable
region (V.sub.LFAP) comprising an amino acid sequence of SEQ ID
NO:10 or of SEQ ID NO:18. More particularly, the VH domain is a
heavy chain variable region (V.sub.HFAP) comprising an amino acid
sequence of SEQ ID NO:17 and the VL domain is a light chain
variable region (V.sub.LFAP) comprising an amino acid sequence of
SEQ ID NO:18.
[0474] In a particular aspect, the invention provides a bispecific
antigen binding molecule comprising [0475] (a) four light chains,
each comprising the amino acid sequence of SEQ ID NO:82, a first
heavy chain comprising the amino acid sequence of SEQ ID NO:83, and
a second heavy chain comprising the amino acid sequence of SEQ ID
NO:84, or [0476] (b) four light chains, each comprising the amino
acid sequence of SEQ ID NO:133, a first heavy chain comprising the
amino acid sequence of SEQ ID NO:131, and a second heavy chain
comprising the amino acid sequence of SEQ ID NO:132.
[0477] In another particular aspect, the invention provides a
bispecific antigen binding molecule capable of specific binding to
murine CD40 comprising [0478] (a) four light chains, each
comprising the amino acid sequence of SEQ ID NO:97, a first heavy
chain comprising the amino acid sequence of SEQ ID NO:95, and a
second heavy chain comprising the amino acid sequence of SEQ ID
NO:96.
[0479] In one aspect, provided is a bispecific antigen binding
molecule, wherein the bispecific antigen binding molecule
comprises
[0480] (a) four light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to
CD40,
[0481] (b) two heavy chains, wherein each of the heavy chain
comprises a VH and CH1 domain of a Fab fragment capable of specific
binding to CD40 fused to a VH and CH1 domain of a second Fab
fragment capable of specific binding to CD40, and a Fc region
subunit, and [0482] (c) one Fab fragment capable of specific
binding to FAP, wherein the Fab fragment is connected to the
C-terminus of one of the two heavy chains of (b).
[0483] In particular, the Fab fragment capable of specific binding
is a crossover fab fragment.
[0484] In one aspect, provided is a bispecific antigen binding
molecule, wherein the bispecific antigen binding molecule
comprises
[0485] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40,
and a Fc region subunit,
[0486] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0487] (c) one Fab fragment capable of specific binding to FAP,
wherein the Fab fragment is connected to the C-terminus of one of
the two heavy chains of (a).
[0488] In particular, the Fab fragment capable of specific binding
is a crossover fab fragment.
[0489] In a further aspect, provided is a bispecific antigen
binding molecule, comprising
[0490] (a) four light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
wherein the VL comprises a light chain variable region
(V.sub.LCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:175, SEQ
ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID
NO:186, SEQ ID NO:187 and SEQ ID NO:188, and
[0491] (b) two heavy chains, each heavy chain comprising a
VH-CH1-VH-CH1 chain and a Fc region subunit, wherein both VH
domains comprise a heavy chain variable region (V.sub.HCD40)
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:171, SEQ ID
NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180,
SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184,
and
[0492] (c) a crossover Fab fragment capable of specific binding to
FAP comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL
chain is connected to the C-terminus of one of the two heavy chains
of (b).
[0493] In one aspect, the VH-CL chain is connected to the
C-terminus of the FC knob heavy chain.
[0494] In a further aspect, provided is a bispecific antigen
binding molecule, comprising
[0495] (a) four light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
wherein the VL comprises a light chain variable region
(V.sub.LCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:175, SEQ
ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID
NO:186, SEQ ID NO:187 and SEQ ID NO:188, and
[0496] (b) two heavy chains, each heavy chain comprising a
VH-CH1-VH-CH1 chain and a Fc region subunit, wherein both VH
domains comprise a heavy chain variable region (V.sub.HCD40)
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:171, SEQ ID
NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180,
SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184,
and
[0497] (c) a crossover fab fragment capable of specific binding to
FAP comprising a VL-CH1 chain and a VH-CL chain, wherein the VL-CH1
chain is connected to the C-terminus of one of the two heavy chains
of (b).
[0498] In one aspect, the VL-CH1 chain is connected to the
C-terminus of the FC knob heavy chain.
[0499] In a particular aspect, the invention provides a bispecific
antigen binding molecule comprising [0500] (a) four light chains,
each comprising the amino acid sequence of SEQ ID NO:165, one light
chain comprising the amino acid sequence of SEQ ID NO:162, a first
heavy chain comprising the amino acid sequence of SEQ ID NO:169,
and a second heavy chain comprising the amino acid sequence of SEQ
ID NO:170, or [0501] (b) two light chains, each comprising the
amino acid sequence of SEQ ID NO:243, one light chain comprising
the amino acid sequence of SEQ ID NO:162, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:244, and a second
heavy chain comprising the amino acid sequence of SEQ ID NO:245, or
[0502] (c) two light chains, each comprising the amino acid
sequence of SEQ ID NO:243, one light chain comprising the amino
acid sequence of SEQ ID NO:162, a first heavy chain comprising the
amino acid sequence of SEQ ID NO:246, and a second heavy chain
comprising the amino acid sequence of SEQ ID NO:247, or [0503] (d)
two light chains, each comprising the amino acid sequence of SEQ ID
NO:248, one light chain comprising the amino acid sequence of SEQ
ID NO:162, a first heavy chain comprising the amino acid sequence
of SEQ ID NO:249, and a second heavy chain comprising the amino
acid sequence of SEQ ID NO:250.
[0504] Bispecific Antigen Binding Molecules Tetravalent for Binding
to CD40 and Bivalent for Binding to the Target Cell Antigen (4+2
format)
[0505] In another aspect, the invention provides a bispecific
antigen binding molecule comprising [0506] (a) four antigen binding
domains capable of specific binding to CD40, [0507] (b) two antigen
binding domains capable of specific binding to a target cell
antigen, and [0508] (c) a Fc domain composed of a first and a
second subunit capable of stable association:
[0509] Thus, provided is a bispecific antigen binding molecule,
wherein the bispecific antigen binding molecule is tetravalent for
CD40 and bivalent for the target cell antigen.
[0510] In one aspect, provided is a bispecific antigen binding
molecule, wherein the four antigen binding domains capable of
specific binding to CD40 are Fab fragments and each two thereof are
fused to each other, optionally via a peptide linker. In a
particular aspect, the peptide linker comprises the amino acid
sequence of SEQ ID NO:148. More particularly, the antigen binding
molecule comprises two heavy chains comprising each a VHCH1-peptide
linker-VHCH1 fragment. In a particular aspect, the peptide linker
has the amino acid sequence of SEQ ID NO:148.
[0511] In another aspect, a bispecific antigen binding molecule is
provided, wherein the antigen binding domains capable of specific
binding to a target cell antigen are Fab fragments and wherein the
first Fab fragment is connected via a peptide linker to the
C-terminus of the first subunit of the Fc domain and the second Fab
fragment is connected via a peptide linker to the C-terminus of the
second subunit of the Fc domain. In one aspect, the two Fab
fragments capable of specific binding to the target cell antigen
are crossover Fab fragments each comprising a VL-CH1 chain and a
VH-CL chain, and wherein the VL-CH1 chain is connected to the
C-terminus of one of the two heavy chains.
[0512] In a particular aspect, the invention provides a bispecific
antigen binding molecule comprising four light chains, each
comprising the amino acid sequence of SEQ ID NO:86, two light
chains, each comprising the amino acid sequence of SEQ ID NO:87,
and two heavy chains comprising the amino acid sequence of SEQ ID
NO:85.
[0513] In another aspect, the invention provides a bispecific
antigen binding molecule capable of specific binding to murine CD40
comprising four light chains, each comprising the amino acid
sequence of SEQ ID NO:100, two light chains, each comprising the
amino acid sequence of SEQ ID NO:99, and two heavy chains
comprising the amino acid sequence of SEQ ID NO:98.
[0514] Fc Domain Modifications Reducing Fc Receptor Binding and/or
Effector Function
[0515] The bispecific antigen binding molecules of the invention
further comprise a Fc domain composed of a first and a second
subunit capable of stable association.
[0516] In certain aspects, one or more amino acid modifications may
be introduced into the Fc region of an antibody provided herein,
thereby generating an Fc region variant. The Fc region variant may
comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3
or IgG4 Fc region) comprising an amino acid modification (e.g. a
substitution) at one or more amino acid positions.
[0517] The Fc domain confers favorable pharmacokinetic properties
to the bispecific antibodies of the invention, including a long
serum half-life which contributes to good accumulation in the
target tissue and a favorable tissue-blood distribution ratio. At
the same time it may, however, lead to undesirable targeting of the
bispecific antibodies of the invention to cells expressing Fc
receptors rather than to the preferred antigen-bearing cells.
Accordingly, in particular embodiments the Fc domain of the
bispecific antibodies of the invention exhibits reduced binding
affinity to an Fc receptor and/or reduced effector function, as
compared to a native IgG Fc domain, in particular an IgG1 Fc domain
or an IgG4 Fc domain. More particularly, the Fc domain is an IgG1
Fc domain.
[0518] In one such aspect the Fc domain (or the bispecific antigen
binding molecule of the invention comprising said Fc domain)
exhibits less than 50%, preferably less than 20%, more preferably
less than 10% and most preferably less than 5% of the binding
affinity to an Fc receptor, as compared to a native IgG1 Fc domain
(or the bispecific antigen binding molecule of the invention
comprising a native IgG1 Fc domain), and/or less than 50%,
preferably less than 20%, more preferably less than 10% and most
preferably less than 5% of the effector function, as compared to a
native IgG1 Fc domain (or the bispecific antigen binding molecule
of the invention comprising a native IgG1 Fc domain). In one
aspect, the Fc domain (or the bispecific antigen binding molecule
of the invention comprising said Fc domain) does not substantially
bind to an Fc receptor and/or induce effector function. In a
particular aspect the Fc receptor is an Fey receptor. In one
aspect, the Fc receptor is a human Fc receptor. In one aspect, the
Fc receptor is an activating Fc receptor. In a specific aspect, the
Fc receptor is an activating human Fc.gamma. receptor, more
specifically human Fc.gamma.RIIIa, Fc.gamma.RI or Fc.gamma.RIIa,
most specifically human Fc.gamma.RIIIa. In one aspect, the Fc
receptor is an inhibitory Fc receptor. In a specific aspect, the Fc
receptor is an inhibitory human Fey receptor, more specifically
human Fc.gamma.RIIB In one aspect the effector function is one or
more of CDC, ADCC, ADCP, and cytokine secretion. In a particular
aspect, the effector function is ADCC. In one aspect, the Fc domain
domain exhibits substantially similar binding affinity to neonatal
Fc receptor (FcRn), as compared to a native IgG1 Fc domain.
Substantially similar binding to FcRn is achieved when the Fc
domain (or the the bispecific antigen binding molecule of the
invention comprising said Fc domain) exhibits greater than about
70%, particularly greater than about 80%, more particularly greater
than about 90% of the binding affinity of a native IgG1 Fc domain
(or the the bispecific antigen binding molecule of the invention
comprising a native IgG1 Fc domain) to FcRn.
[0519] In a particular aspect, the Fc domain is engineered to have
reduced binding affinity to an Fc receptor and/or reduced effector
function, as compared to a non-engineered Fc domain. In a
particular aspect, the Fc domain of the bispecific antigen binding
molecule of the invention comprises one or more amino acid mutation
that reduces the binding affinity of the Fc domain to an Fc
receptor and/or effector function. Typically, the same one or more
amino acid mutation is present in each of the two subunits of the
Fc domain. In one aspect, the amino acid mutation reduces the
binding affinity of the Fc domain to an Fc receptor. In another
aspect, the amino acid mutation reduces the binding affinity of the
Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or
at least 10-fold. In one aspect, the bispecific antigen binding
molecule of the invention comprising an engineered Fc domain
exhibits less than 20%, particularly less than 10%, more
particularly less than 5% of the binding affinity to an Fc receptor
as compared to bispecific antibodies of the invention comprising a
non-engineered Fc domain. In a particular aspect, the Fc receptor
is an Fc.gamma. receptor. In other aspects, the Fc receptor is a
human Fc receptor. In one aspect, the Fc receptor is an inhibitory
Fc receptor. In a specific aspect, the Fc receptor is an inhibitory
human Fey receptor, more specifically human Fc.gamma.RIIB In some
aspects the Fc receptor is an activating Fc receptor. In a specific
aspect, the Fc receptor is an activating human Fc.gamma. receptor,
more specifically human Fc.gamma.RIIIa, Fc.gamma.RI or
Fc.gamma.RIIa, most specifically human Fc.gamma.RIIIa. Preferably,
binding to each of these receptors is reduced. In some aspects,
binding affinity to a complement component, specifically binding
affinity to C1q, is also reduced. In one aspect, binding affinity
to neonatal Fc receptor (FcRn) is not reduced.
[0520] Substantially similar binding to FcRn, i.e. preservation of
the binding affinity of the Fc domain to said receptor, is achieved
when the Fc domain (or the bispecific antigen binding molecule of
the invention comprising said Fc domain) exhibits greater than
about 70% of the binding affinity of a non-engineered form of the
Fc domain (or the bispecific antigen binding molecule of the
invention comprising said non-engineered form of the Fc domain) to
FcRn. The Fc domain, or the the bispecific antigen binding molecule
of the invention comprising said Fc domain, may exhibit greater
than about 80% and even greater than about 90% of such affinity. In
certain embodiments the Fc domain of the bispecific antigen binding
molecule of the invention is engineered to have reduced effector
function, as compared to a non-engineered Fc domain. The reduced
effector function can include, but is not limited to, one or more
of the following: reduced complement dependent cytotoxicity (CDC),
reduced antibody-dependent cell-mediated cytotoxicity (ADCC),
reduced antibody-dependent cellular phagocytosis (ADCP), reduced
cytokine secretion, reduced immune complex-mediated antigen uptake
by antigen-presenting cells, reduced binding to NK cells, reduced
binding to macrophages, reduced binding to monocytes, reduced
binding to polymorphonuclear cells, reduced direct signaling
inducing apoptosis, reduced dendritic cell maturation, or reduced T
cell priming.
[0521] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581). Certain antibody variants with
improved or diminished binding to FcRs are described. (e.g. U.S.
Pat. No. 6,737,056; WO 2004/056312, and Shields, R. L. et al., J.
Biol. Chem. 276 (2001) 6591-6604).
[0522] In one aspect of the invention, the Fc domain comprises an
amino acid substitution at a position of E233, L234, L235, N297,
P331 and P329. In some aspects, the Fc domain comprises the amino
acid substitutions L234A and L235A ("LALA"). In one such
embodiment, the Fc domain is an IgG1 Fc domain, particularly a
human IgG1 Fc domain. In one aspect, the Fc domain comprises an
amino acid substitution at position P329. In a more specific
aspect, the amino acid substitution is P329A or P329G, particularly
P329G. In one embodiment the Fc domain comprises an amino acid
substitution at position P329 and a further amino acid substitution
selected from the group consisting of E233P, L234A, L235A, L235E,
N297A, N297D or P331S. In more particular embodiments the Fc domain
comprises the amino acid mutations L234A, L235A and P329G ("P329G
LALA"). The "P329G LALA" combination of amino acid substitutions
almost completely abolishes Fc.gamma. receptor binding of a human
IgG1 Fc domain, as described in PCT Patent Application No. WO
2012/130831 Al. Said document also describes methods of preparing
such mutant Fc domains and methods for determining its properties
such as Fc receptor binding or effector functions. Such antibody is
an IgG1 with mutations L234A and L235A or with mutations L234A,
L235A and P329G (numbering according to EU index of Kabat et al.,
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md., 1991).
[0523] In one aspect, the Fc domain is an IgG4 Fc domain. In a more
specific embodiment, the Fc domain is an IgG4 Fc domain comprising
an amino acid substitution at position 5228 (Kabat numbering),
particularly the amino acid substitution S228P. In a more specific
embodiment, the Fc domain is an IgG4 Fc domain comprising amino
acid substitutions L235E and S228P and P329G. This amino acid
substitution reduces in vivo Fab arm exchange of IgG4 antibodies
(see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91
(2010)).
[0524] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer, R. L. et al., J.
Immunol. 117 (1976) 587-593, and Kim, J. K. et al., J. Immunol. 24
(1994) 2429-2434), are described in US 2005/0014934. Those
antibodies comprise an Fc region with one or more substitutions
therein which improve binding of the Fc region to FcRn. Such Fc
variants include those with substitutions at one or more of Fc
region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,
317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g.,
substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).
See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740;
U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0525] Binding to Fc receptors can be easily determined e.g. by
ELISA, or by Surface Plasmon Resonance (SPR) using standard
instrumentation such as a BlAcore instrument (GE Healthcare), and
Fc receptors such as may be obtained by recombinant expression. A
suitable such binding assay is described herein. Alternatively,
binding affinity of Fc domains or cell activating bispecific
antigen binding molecules comprising an Fc domain for Fc receptors
may be evaluated using cell lines known to express particular Fc
receptors, such as human NK cells expressing Fc.gamma.IIIa
receptor. Effector function of an Fc domain, or bispecific antigen
binding molecules of the invention comprising an Fc domain, can be
measured by methods known in the art. A suitable assay for
measuring ADCC is described herein. Other examples of in vitro
assays to assess ADCC activity of a molecule of interest are
described in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl
Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl
Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;
Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,
non-radioactive assays methods may be employed (see, for example,
ACTI.TM. non-radioactive cytotoxicity assay for flow cytometry
(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Madison, Wis.)).
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g. in a animal model such as
that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656
(1998).
[0526] The following section describes preferred aspects of the
bispecific antigen binding molecules of the invention comprising Fc
domain modifications reducing Fc receptor binding and/or effector
function. In one aspect, the invention relates to the bispecific
antigen binding molecule (a) at least one antigen binding domain
capable of specific binding to CD40, (b) at least one antigen
binding domain capable of specific binding to a target cell
antigen, and (c) a Fc domain composed of a first and a second
subunit capable of stable association, wherein the Fc domain
comprises one or more amino acid substitution that reduces the
binding affinity of the antibody to an Fc receptor, in particular
towards Fc.gamma. receptor. In another aspect, the invention
relates to the bispecific antigen binding molecule comprising (a)
at least one antigen binding domain capable of specific binding to
CD40, (b) at least one antigen binding domain capable of specific
binding to a target cell antigen, and (c) a Fc domain composed of a
first and a second subunit capable of stable association, wherein
the Fc domain comprises one or more amino acid substitution that
reduces effector function. In particular aspect, the Fc domain is
of human IgG1 subclass with the amino acid mutations L234A, L235A
and P329G (numbering according to Kabat EU index).
[0527] Fc Domain Modifications Promoting Heterodimerization
[0528] The bispecific antigen binding molecules of the invention
comprise different antigen-binding sites, fused to one or the other
of the two subunits of the Fc domain, thus the two subunits of the
Fc domain may be comprised in two non-identical polypeptide chains.
Recombinant co-expression of these polypeptides and subsequent
dimerization leads to several possible combinations of the two
polypeptides. To improve the yield and purity of the bispecific
antigen binding molecules of the invention in recombinant
production, it will thus be advantageous to introduce in the Fc
domain of the bispecific antigen binding molecules of the invention
a modification promoting the association of the desired
polypeptides.
[0529] Accordingly, in particular aspects the invention relates to
the bispecific antigen binding molecule comprising (a) at least one
antigen binding domain capable of specific binding to CD40, (b) at
least one antigen binding domain capable of specific binding to a
target cell antigen, and (c) a Fc domain composed of a first and a
second subunit capable of stable association, wherein the Fc domain
comprises a modification promoting the association of the first and
second subunit of the Fc domain. The site of most extensive
protein-protein interaction between the two subunits of a human IgG
Fc domain is in the CH3 domain of the Fc domain. Thus, in one
aspect said modification is in the CH3 domain of the Fc domain.
[0530] In a specific aspect said modification is a so-called
"knob-into-hole" modification, comprising a "knob" modification in
one of the two subunits of the Fc domain and a "hole" modification
in the other one of the two subunits of the Fc domain. Thus, the
invention relates to the bispecific antigen binding molecule
comprising (a) at least one antigen binding domain capable of
specific binding to CD40, (b) at least one antigen binding domain
capable of specific binding to a target cell antigen, and (c) a Fc
domain composed of a first and a second subunit capable of stable
association, wherein the first subunit of the Fc domain comprises
knobs and the second subunit of the Fc domain comprises holes
according to the knobs into holes method. In a particular aspect,
the first subunit of the Fc domain comprises the amino acid
substitutions S354C and T366W (EU numbering) and the second subunit
of the Fc domain comprises the amino acid substitutions Y349C,
T366S and Y407V (numbering according to Kabat EU index).
[0531] The knob-into-hole technology is described e.g. in U.S. Pat.
No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9,
617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
Generally, the method involves introducing a protuberance ("knob")
at the interface of a first polypeptide and a corresponding cavity
("hole") in the interface of a second polypeptide, such that the
protuberance can be positioned in the cavity so as to promote
heterodimer formation and hinder homodimer formation. Protuberances
are constructed by replacing small amino acid side chains from the
interface of the first polypeptide with larger side chains (e.g.
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino acid side chains
with smaller ones (e.g. alanine or threonine).
[0532] Accordingly, in one aspect, in the CH3 domain of the first
subunit of the Fc domain of the bispecific antigen binding
molecules of the invention an amino acid residue is replaced with
an amino acid residue having a larger side chain volume, thereby
generating a protuberance within the CH3 domain of the first
subunit which is positionable in a cavity within the CH3 domain of
the second subunit, and in the CH3 domain of the second subunit of
the Fc domain an amino acid residue is replaced with an amino acid
residue having a smaller side chain volume, thereby generating a
cavity within the CH3 domain of the second subunit within which the
protuberance within the CH3 domain of the first subunit is
positionable. The protuberance and cavity can be made by altering
the nucleic acid encoding the polypeptides, e.g. by site-specific
mutagenesis, or by peptide synthesis. In a specific aspect, in the
CH3 domain of the first subunit of the Fc domain the threonine
residue at position 366 is replaced with a tryptophan residue
(T366W), and in the CH3 domain of the second subunit of the Fc
domain the tyrosine residue at position 407 is replaced with a
valine residue (Y407V). In one aspect, in the second subunit of the
Fc domain additionally the threonine residue at position 366 is
replaced with a serine residue (T366S) and the leucine residue at
position 368 is replaced with an alanine residue (L368A).
[0533] In yet a further aspect, in the first subunit of the Fc
domain additionally the serine residue at position 354 is replaced
with a cysteine residue (S354C), and in the second subunit of the
Fc domain additionally the tyrosine residue at position 349 is
replaced by a cysteine residue (Y349C). Introduction of these two
cysteine residues results in formation of a disulfide bridge
between the two subunits of the Fc domain, further stabilizing the
dimer (Carter (2001), J Immunol Methods 248, 7-15). In a particular
aspect, the first subunit of the Fc domain comprises the amino acid
substitutions S354C and T366W (EU numbering) and the second subunit
of the Fc domain comprises the amino acid substitutions Y349C,
T366S and Y407V (numbering according to Kabat EU index).
[0534] In an alternative aspect, a modification promoting
association of the first and the second subunit of the Fc domain
comprises a modification mediating electrostatic steering effects,
e.g. as described in PCT publication WO 2009/089004. Generally,
this method involves replacement of one or more amino acid residues
at the interface of the two Fc domain subunits by charged amino
acid residues so that homodimer formation becomes electrostatically
unfavorable but heterodimerization electrostatically favorable.
[0535] The C-terminus of the heavy chain of the bispecific antibody
as reported herein can be a complete C-terminus ending with the
amino acid residues PGK. The C-terminus of the heavy chain can be a
shortened C-terminus in which one or two of the C terminal amino
acid residues have been removed. In one preferred aspect, the
C-terminus of the heavy chain is a shortened C-terminus ending PG.
In one aspect of all aspects as reported herein, a bispecific
antibody comprising a heavy chain including a C-terminal CH3 domain
as specified herein, comprises the C-terminal glycine-lysine
dipeptide (G446 and K447, numbering according to Kabat EU index).
In one embodiment of all aspects as reported herein, a bispecific
antibody comprising a heavy chain including a C-terminal CH3
domain, as specified herein, comprises a C-terminal glycine residue
(G446, numbering according to Kabat EU index).
[0536] Modifications in the Fab Domains
[0537] In one aspect, the invention relates to a bispecific antigen
binding molecule comprising (a) a first Fab fragment capable of
specific binding to CD40, (b) a second Fab fragment capable of
specific binding to a target cell antigen, and (c) a Fc domain
composed of a first and a second subunit capable of stable
association, wherein in one of the Fab fragments either the
variable domains VH and VL or the constant domains CH1 and CL are
exchanged. The bispecific antibodies are prepared according to the
Crossmab technology.
[0538] Multispecific antibodies with a domain replacement/exchange
in one binding arm (CrossMabVH-VL or CrossMabCH-CL) are described
in detail in WO2009/080252 and Schaefer, W. et al, PNAS, 108 (2011)
11187-1191. They clearly reduce the byproducts caused by the
mismatch of a light chain against a first antigen with the wrong
heavy chain against the second antigen (compared to approaches
without such domain exchange).
[0539] In one aspect, the invention relates to a bispecific antigen
binding molecule comprising (a) a first Fab fragment capable of
specific binding to CD40, (b) a second Fab fragment capable of
specific binding to a target cell antigen, and (c) a Fc domain
composed of a first and a second subunit capable of stable
association, wherein in one of the Fab fragments the constant
domains CL and CH1 are replaced by each other so that the CH1
domain is part of the light chain and the CL domain is part of the
heavy chain. More particularly, in the second Fab fragment capable
of specific binding to a target cell antigen the constant domains
CL and CH1 are replaced by each other so that the CH1 domain is
part of the light chain and the CL domain is part of the heavy
chain.
[0540] In a particular aspect, the invention relates a bispecific
antigen binding molecule comprising (a) a first Fab fragment
capable of specific binding to CD40, (b) a second Fab fragment
capable of specific binding to a target cell antigen, wherein the
constant domains CL and CH1 are replaced by each other so that the
CH1 domain is part of the light chain and the CL domain is part of
the heavy chain. Such a molecule is called a monovalent bispecific
antigen binding molecule.
[0541] In another aspect, the invention relates to a bispecific
antigen binding molecule, comprising (a) two light chains and two
heavy chains of an antibody comprising two Fab fragments capable of
specific binding to CD40 and the Fc domain, and (b) two additional
Fab fragments capable of specific binding to a target cell antigen,
wherein said additional Fab fragments are each connected via a
peptide linker to the C-terminus of the heavy chains of (a). In a
particular aspect, the additional Fab fragments are Fab fragments,
wherein the variable domains VL and VH are replaced by each other
so that the VH domain is part of the light chain and the VL domain
is part of the heavy chain.
[0542] Thus, in a particular aspect, the invention comprises a
bispecific antigen binding molecule, comprising (a) two light
chains and two heavy chains of an antibody comprising two Fab
fragments capable of specific binding to CD40 and the Fc domain,
and (b) two additional Fab fragments capable of specific binding to
a target cell antigen, wherein said two additional Fab fragments
capable of specific binding to a target cell antigen are crossover
Fab fragments wherein the variable domains VL and VH are replaced
by each other and the VL-CH chains are each connected via a peptide
linker to the C-terminus of the heavy chains of (a).
[0543] In another aspect, and to further improve correct pairing,
the bispecific antigen binding molecule comprising (a) a first Fab
fragment capable of specific binding to CD40, (b) a second Fab
fragment capable of specific binding to a target cell antigen, and
(c) a Fc domain composed of a first and a second subunit capable of
stable association, can contain different charged amino acid
substitutions (so-called "charged residues"). These modifications
are introduced in the crossed or non-crossed CH1 and CL domains. In
a particular aspect, the invention relates to a bispecific antigen
binding molecule, wherein in one of CL domains the amino acid at
position 123 (EU numbering) has been replaced by arginine (R) and
the amino acid at position 124 (EU numbering) has been substituted
by lysine (K) and wherein in one of the CH1 domains the amino acids
at position 147 (EU numbering) and at position 213 (EU numbering)
have been substituted by glutamic acid (E).
[0544] Exemplary Antibodies of the Invention
[0545] In one aspect, the invention provides new antibodies and
antibody fragments that specifically bind to CD40. These antibodies
have superior properties compared to the known CD40 antibodies that
make them especially suitable for the incorporation into bispecific
antigen binding molecules comprising another antigen binding moiety
capable of specific binding to a target cell antigen. The new
antibodies are further characterized in that they are producable in
high amounts and with high titers, that they show high thermal
stability (as measured by the aggregation temperature T.sub.agg),
or in that they possess a high degree of humanness and may
therefore be less immunogenic in the human body. The percentage of
humanness of the VH and VL sequences as compared to the human
germline sequences can be determined by the methods described in
Abhinandan, K. R. and Martin, Andrew C. R. 2007, J. Mol. Biol.
2007, 369, 852-862. The corresponding data are shown in Tables 24
and 25.
[0546] In one aspect, provided is an antibody that specifically
binds to CD40, wherein said antibody comprises [0547] (i) a heavy
chain variable region VH comprising an amino acid sequence of SEQ
ID NO:171 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:175, [0548] (ii) a heavy chain variable
region VH comprising an amino acid sequence of SEQ ID NO:173 and a
light chain variable region VL comprising an amino acid sequence of
SEQ ID NO:177, [0549] (iii) a heavy chain variable region VH
comprising an amino acid sequence of SEQ ID NO:174 and a light
chain variable region VL comprising an amino acid sequence of SEQ
ID NO:178, [0550] (iv) a heavy chain variable region VH comprising
an amino acid sequence of SEQ ID NO:171 and a light chain variable
region VL comprising an amino acid sequence of SEQ ID NO:177,
[0551] (v) a heavy chain variable region VH comprising an amino
acid sequence of SEQ ID NO:171 and a light chain variable region VL
comprising an amino acid sequence of SEQ ID NO:178, [0552] (vi) a
heavy chain variable region VH comprising an amino acid sequence of
SEQ ID NO:173 and a light chain variable region VL comprising an
amino acid sequence of SEQ ID NO:175, or [0553] (vii) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:173 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:178, or [0554] (viii) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:174 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:175, or [0555] (viii) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:174 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:177, or [0556] (ix) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:171 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:176, or [0557] (x) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:172 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:175, or [0558] (xi) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:172 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:176, or [0559] (xii) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:172 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:177, or [0560] (xiii) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:172 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:178, or [0561] (xiv) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:173 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:176, or [0562] (xv) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:174 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:176.
[0563] In another aspect, provided is an antibody that competes for
binding with an antibody that specifically binds to CD40, wherein
said antibody comprises any of the heavy chain variable region VH
and a light chain variable region VL of (i) to (xv) as defined
herein before.
[0564] In one aspect, provided is an antibody that competes for
binding with an antibody that specifically binds to CD40, wherein
said antibody comprises a heavy chain variable region VH comprising
an amino acid sequence of SEQ ID NO:171 and a light chain variable
region VL comprising an amino acid sequence of SEQ ID NO:175.
[0565] In a further aspect, provided is an antibody that
specifically binds to CD40, wherein said antibody comprises [0566]
(i) a heavy chain variable region VH comprising an amino acid
sequence of SEQ ID NO:179 and a light chain variable region VL
comprising an amino acid sequence of SEQ ID NO:185, [0567] (ii) a
heavy chain variable region VH comprising an amino acid sequence of
SEQ ID NO:180 and a light chain variable region VL comprising an
amino acid sequence of SEQ ID NO:185, [0568] (iii) a heavy chain
variable region VH comprising an amino acid sequence of SEQ ID
NO:181 and a light chain variable region VL comprising an amino
acid sequence of SEQ ID NO:185, [0569] (iv) a heavy chain variable
region VH comprising an amino acid sequence of SEQ ID NO:182 and a
light chain variable region VL comprising an amino acid sequence of
SEQ ID NO:185, [0570] (v) a heavy chain variable region VH
comprising an amino acid sequence of SEQ ID NO:179 and a light
chain variable region VL comprising an amino acid sequence of SEQ
ID NO:186, [0571] (vi) a heavy chain variable region VH comprising
an amino acid sequence of SEQ ID NO:180 and a light chain variable
region VL comprising an amino acid sequence of SEQ ID NO:186, or
[0572] (vii) a heavy chain variable region VH comprising an amino
acid sequence of SEQ ID NO:181 and a light chain variable region VL
comprising an amino acid sequence of SEQ ID NO:186, or [0573]
(viii) a heavy chain variable region VH comprising an amino acid
sequence of SEQ ID NO:1182 and a light chain variable region VL
comprising an amino acid sequence of SEQ ID NO:186.
[0574] In another aspect, provided is an antibody that competes for
binding with an antibody that specifically binds to CD40, wherein
said antibody comprises any of the heavy chain variable region VH
and a light chain variable region VL of (i) to (viii) as defined
herein before.
[0575] In one aspect, provided is an antibody that competes for
binding with an antibody that specifically binds to CD40, wherein
said antibody comprises a heavy chain variable region VH comprising
an amino acid sequence of SEQ ID NO:179 and a light chain variable
region VL comprising an amino acid sequence of SEQ ID NO:185. In
particular, provided is an antibody that specifically binds to
CD40, wherein said antibody comprises a heavy chain variable region
VH comprising an amino acid sequence of SEQ ID NO:182 and and a
light chain variable region VL comprising an amino acid sequence of
SEQ ID NO:185.
[0576] Polynucleotides
[0577] The invention further provides isolated polynucleotides
encoding a bispecific antigen binding molecule as described herein
or a fragment thereof or polynucleotides encoding an antibody as
described herein.
[0578] The isolated polynucleotides encoding bispecific antigen
binding molecules of the invention may be expressed as a single
polynucleotide that encodes the entire antigen binding molecule or
as multiple (e.g., two or more) polynucleotides that are
co-expressed. Polypeptides encoded by polynucleotides that are
co-expressed may associate through, e.g., disulfide bonds or other
means to form a functional antigen binding molecule. For example,
the light chain portion of an immunoglobulin may be encoded by a
separate polynucleotide from the heavy chain portion of the
immunoglobulin. When co-expressed, the heavy chain polypeptides
will associate with the light chain polypeptides to form the
immunoglobulin.
[0579] In some aspects, the isolated polynucleotide encodes a
polypeptide comprised in the bispecific molecule according to the
invention as described herein.
[0580] In one aspect, the present invention is directed to an
isolated polynucleotide encoding a bispecific antigen binding
molecule, comprising (a) at least one antigen binding domain
capable of specific binding to CD40, (b) at least one antigen
binding domain capable of specific binding to a target cell
antigen, and (c) a Fc domain composed of a first and a second
subunit capable of stable association.
[0581] In certain embodiments the polynucleotide or nucleic acid is
DNA. In other embodiments, a polynucleotide of the present
invention is RNA, for example, in the form of messenger RNA (mRNA).
RNA of the present invention may be single stranded or double
stranded.
[0582] Recombinant Methods
[0583] Bispecific antigen binding molecules of the invention may be
obtained, for example, by recombinant production. For recombinant
production one or more polynucleotide encoding the bispecific
antigen binding molecule or polypeptide fragments thereof are
provided. The one or more polynucleotide encoding the bispecific
antigen binding molecule are isolated and inserted into one or more
vectors for further cloning and/or expression in a host cell. Such
polynucleotide may be readily isolated and sequenced using
conventional procedures. In one aspect of the invention, a vector,
preferably an expression vector, comprising one or more of the
polynucleotides of the invention is provided. Methods which are
well known to those skilled in the art can be used to construct
expression vectors containing the coding sequence of the bispecific
antigen binding molecule (fragment) along with appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination/genetic recombination. See, for example,
the techniques described in Maniatis et al., MOLECULAR CLONING: A
LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene
Publishing Associates and Wiley Interscience, N.Y. (1989). The
expression vector can be part of a plasmid, virus, or may be a
nucleic acid fragment. The expression vector includes an expression
cassette into which the polynucleotide encoding the bispecific
antigen binding molecule or polypeptide fragments thereof (i.e. the
coding region) is cloned in operable association with a promoter
and/or other transcription or translation control elements. As used
herein, a "coding region" is a portion of nucleic acid which
consists of codons translated into amino acids. Although a "stop
codon" (TAG, TGA, or TAA) is not translated into an amino acid, it
may be considered to be part of a coding region, if present, but
any flanking sequences, for example promoters, ribosome binding
sites, transcriptional terminators, introns, 5' and 3' untranslated
regions, and the like, are not part of a coding region. Two or more
coding regions can be present in a single polynucleotide construct,
e.g. on a single vector, or in separate polynucleotide constructs,
e.g. on separate (different) vectors. Furthermore, any vector may
contain a single coding region, or may comprise two or more coding
regions, e.g. a vector of the present invention may encode one or
more polypeptides, which are post- or co-translationally separated
into the final proteins via proteolytic cleavage. In addition, a
vector, polynucleotide, or nucleic acid of the invention may encode
heterologous coding regions, either fused or unfused to a
polynucleotide encoding the bispecific antigen binding molecule of
the invention or polypeptide fragments thereof, or variants or
derivatives thereof. Heterologous coding regions include without
limitation specialized elements or motifs, such as a secretory
signal peptide or a heterologous functional domain. An operable
association is when a coding region for a gene product, e.g. a
polypeptide, is associated with one or more regulatory sequences in
such a way as to place expression of the gene product under the
influence or control of the regulatory sequence(s). Two DNA
fragments (such as a polypeptide coding region and a promoter
associated therewith) are "operably associated" if induction of
promoter function results in the transcription of mRNA encoding the
desired gene product and if the nature of the linkage between the
two DNA fragments does not interfere with the ability of the
expression regulatory sequences to direct the expression of the
gene product or interfere with the ability of the DNA template to
be transcribed. Thus, a promoter region would be operably
associated with a nucleic acid encoding a polypeptide if the
promoter was capable of effecting transcription of that nucleic
acid. The promoter may be a cell-specific promoter that directs
substantial transcription of the DNA only in predetermined cells.
Other transcription control elements, besides a promoter, for
example enhancers, operators, repressors, and transcription
termination signals, can be operably associated with the
polynucleotide to direct cell-specific transcription.
[0584] Suitable promoters and other transcription control regions
are disclosed herein. A variety of transcription control regions
are known to those skilled in the art. These include, without
limitation, transcription control regions, which function in
vertebrate cells, such as, but not limited to, promoter and
enhancer segments from cytomegaloviruses (e.g. the immediate early
promoter, in conjunction with intron-A), simian virus 40 (e.g. the
early promoter), and retroviruses (such as, e.g. Rous sarcoma
virus). Other transcription control regions include those derived
from vertebrate genes such as actin, heat shock protein, bovine
growth hormone and rabbit a-globin, as well as other sequences
capable of controlling gene expression in eukaryotic cells.
Additional suitable transcription control regions include
tissue-specific promoters and enhancers as well as inducible
promoters (e.g. promoters inducible tetracyclins). Similarly, a
variety of translation control elements are known to those of
ordinary skill in the art. These include, but are not limited to
ribosome binding sites, translation initiation and termination
codons, and elements derived from viral systems (particularly an
internal ribosome entry site, or IRES, also referred to as a CITE
sequence). The expression cassette may also include other features
such as an origin of replication, and/or chromosome integration
elements such as retroviral long terminal repeats (LTRs), or
adeno-associated viral (AAV) inverted terminal repeats (ITRs).
[0585] Polynucleotide and nucleic acid coding regions of the
present invention may be associated with additional coding regions
which encode secretory or signal peptides, which direct the
secretion of a polypeptide encoded by a polynucleotide of the
present invention. For example, if secretion of the bispecific
antigen binding molecule or polypeptide fragments thereof is
desired, DNA encoding a signal sequence may be placed upstream of
the nucleic acid encoding the bispecific antigen binding molecule
of the invention or polypeptide fragments thereof. According to the
signal hypothesis, proteins secreted by mammalian cells have a
signal peptide or secretory leader sequence which is cleaved from
the mature protein once export of the growing protein chain across
the rough endoplasmic reticulum has been initiated. Those of
ordinary skill in the art are aware that polypeptides secreted by
vertebrate cells generally have a signal peptide fused to the
N-terminus of the polypeptide, which is cleaved from the translated
polypeptide to produce a secreted or "mature" form of the
polypeptide. In certain embodiments, the native signal peptide,
e.g. an immunoglobulin heavy chain or light chain signal peptide is
used, or a functional derivative of that sequence that retains the
ability to direct the secretion of the polypeptide that is operably
associated with it. Alternatively, a heterologous mammalian signal
peptide, or a functional derivative thereof, may be used. For
example, the wild-type leader sequence may be substituted with the
leader sequence of human tissue plasminogen activator (TPA) or
mouse .beta.-glucuronidase.
[0586] DNA encoding a short protein sequence that could be used to
facilitate later purification (e.g. a histidine tag) or assist in
labeling the fusion protein may be included within or at the ends
of the polynucleotide encoding a bispecific antigen binding
molecule of the invention or polypeptide fragments thereof.
[0587] In a further aspect of the invention, a host cell comprising
one or more polynucleotides of the invention is provided. In
certain aspects, a host cell comprising one or more vectors of the
invention is provided. The polynucleotides and vectors may
incorporate any of the features, singly or in combination,
described herein in relation to polynucleotides and vectors,
respectively. In one aspect, a host cell comprises (e.g. has been
transformed or transfected with) a vector comprising a
polynucleotide that encodes (part of) a bispecific antigen binding
molecule of the invention of the invention. As used herein, the
term "host cell" refers to any kind of cellular system which can be
engineered to generate the fusion proteins of the invention or
fragments thereof. Host cells suitable for replicating and for
supporting expression of antigen binding molecules are well known
in the art. Such cells may be transfected or transduced as
appropriate with the particular expression vector and large
quantities of vector containing cells can be grown for seeding
large scale fermenters to obtain sufficient quantities of the
antigen binding molecule for clinical applications. Suitable host
cells include prokaryotic microorganisms, such as E. coli, or
various eukaryotic cells, such as Chinese hamster ovary cells
(CHO), insect cells, or the like. For example, polypeptides may be
produced in bacteria in particular when glycosylation is not
needed. After expression, the polypeptide may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified. In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for polypeptide-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized", resulting in
the production of a polypeptide with a partially or fully human
glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414
(2004), and Li et al., Nat Biotech 24, 210-215 (2006).
[0588] Suitable host cells for the expression of (glycosylated)
polypeptides are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells. Plant
cell cultures can also be utilized as hosts. See e.g. U.S. Pat.
Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429
(describing PLANTIBODIES.TM. technology for producing antibodies in
transgenic plants). Vertebrate cells may also be used as hosts. For
example, mammalian cell lines that are adapted to grow in
suspension may be useful. Other examples of useful mammalian host
cell lines are monkey kidney CV1 line transformed by SV40 (COS-7);
human embryonic kidney line (293 or 293T cells as described, e.g.,
in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney
cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in
Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1),
African green monkey kidney cells (VERO-76), human cervical
carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat
liver cells (BRL 3A), human lung cells (W138), human liver cells
(Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as
described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68
(1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host
cell lines include Chinese hamster ovary (CHO) cells, including
dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216
(1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.
For a review of certain mammalian host cell lines suitable for
protein production, see, e.g., Yazaki and Wu, Methods in Molecular
Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.),
pp. 255-268 (2003). Host cells include cultured cells, e.g.,
mammalian cultured cells, yeast cells, insect cells, bacterial
cells and plant cells, to name only a few, but also cells comprised
within a transgenic animal, transgenic plant or cultured plant or
animal tissue. In one embodiment, the host cell is a eukaryotic
cell, preferably a mammalian cell, such as a Chinese Hamster Ovary
(CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell
(e.g., Y0, NS0, Sp20 cell). Standard technologies are known in the
art to express foreign genes in these systems. Cells expressing a
polypeptide comprising either the heavy or the light chain of an
immunoglobulin, may be engineered so as to also express the other
of the immunoglobulin chains such that the expressed product is an
immunoglobulin that has both a heavy and a light chain.
[0589] In one aspect, a method of producing a bispecific antigen
binding molecule of the invention or polypeptide fragments thereof
is provided, wherein the method comprises culturing a host cell
comprising polynucleotides encoding the bispecific antigen binding
molecule of the invention or polypeptide fragments thereof, as
provided herein, under conditions suitable for expression of the
bispecific antigen binding molecule of the invention or polypeptide
fragments thereof, and recovering the bispecific antigen binding
molecule of the invention or polypeptide fragments thereof from the
host cell (or host cell culture medium).
[0590] Bispecific molecules of the invention prepared as described
herein may be purified by art-known techniques such as high
performance liquid chromatography, ion exchange chromatography, gel
electrophoresis, affinity chromatography, size exclusion
chromatography, and the like. The actual conditions used to purify
a particular protein will depend, in part, on factors such as net
charge, hydrophobicity, hydrophilicity etc., and will be apparent
to those having skill in the art. For affinity chromatography
purification an antibody, ligand, receptor or antigen can be used
to which the bispecific antigen binding molecule binds. For
example, for affinity chromatography purification of fusion
proteins of the invention, a matrix with protein A or protein G may
be used. Sequential Protein A or G affinity chromatography and size
exclusion chromatography can be used to isolate an antigen binding
molecule essentially as described in the examples. The purity of
the bispecific antigen binding molecule or fragments thereof can be
determined by any of a variety of well-known analytical methods
including gel electrophoresis, high pressure liquid chromatography,
and the like. For example, the bispecific antigen binding molecules
expressed as described in the Examples were shown to be intact and
properly assembled as demonstrated by reducing and non-reducing
SDS-PAGE.
[0591] Assays
[0592] The antigen binding molecules provided herein may be
characterized for their binding properties and/or biological
activity by various assays known in the art. In particular, they
are characterized by the assays described in more detail in the
examples.
[0593] 1. Binding Assay
[0594] Binding of the bispecific antigen binding molecule provided
herein to the corresponding target expressing cells may be
evaluated for example by using a murine fibroblast cell line
expressing human Fibroblast Activation Protein (FAP) and flow
cytometry (FACS) analysis. Binding of the bispecific antigen
binding molecules provided herein to CD40 may be determined by
using Raji cells as described in Example 4.2.8.
[0595] 2. Activity Assays
[0596] Bispecific antigen binding molecules of the invention are
tested for biological activity. Biological activity may include
efficacy and specificity of the bispecific antigen binding
molecules. Efficacy and specificity are demonstrated by assays
showing agonistic signaling through the CD40 receptor upon binding
of the target antigen. Furthermore in vitro T cell priming assays
are conducted using dendritic cells (DCs) that have been incubated
with the bispecific antigen binding molecules.
[0597] Pharmaceutical Compositions, Formulations and Routes of
Administation
[0598] In a further aspect, the invention provides pharmaceutical
compositions comprising any of the bispecific antigen binding
molecules provided herein, e.g., for use in any of the below
therapeutic methods. In one embodiment, a pharmaceutical
composition comprises any of the bispecific antigen binding
molecules provided herein and at least one pharmaceutically
acceptable excipient. In another embodiment, a pharmaceutical
composition comprises any of the bispecific antigen binding
molecules provided herein and at least one additional therapeutic
agent, e.g., as described below.
[0599] Pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of one or more
bispecific antigen binding molecules dissolved or dispersed in a
pharmaceutically acceptable excipient. The phrases "pharmaceutical
or pharmacologically acceptable" refers to molecular entities and
compositions that are generally non-toxic to recipients at the
dosages and concentrations employed, i.e. do not produce an
adverse, allergic or other untoward reaction when administered to
an animal, such as, for example, a human, as appropriate. The
preparation of a pharmaceutical composition that contains at least
one bispecific antigen binding molecule according to the invention
and optionally an additional active ingredient will be known to
those of skill in the art in light of the present disclosure, as
exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack
Printing Company, 1990, incorporated herein by reference. In
particular, the compositions are lyophilized formulations or
aqueous solutions. As used herein, "pharmaceutically acceptable
excipient" includes any and all solvents, buffers, dispersion
media, coatings, surfactants, antioxidants, preservatives (e.g.
antibacterial agents, antifungal agents), isotonic agents, salts,
stabilizers and combinations thereof, as would be known to one of
ordinary skill in the art.
[0600] Parenteral compositions include those designed for
administration by injection, e.g. subcutaneous, intradermal,
intralesional, intravenous, intraarterial intramuscular,
intrathecal or intraperitoneal injection. For injection, the
bispecific antigen binding molecules of the invention may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer. The solution may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the bispecific antigen binding molecules may be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use. Sterile injectable solutions are
prepared by incorporating the antigen binding molecules of the
invention in the required amount in the appropriate solvent with
various of the other ingredients enumerated below, as required.
Sterility may be readily accomplished, e.g., by filtration through
sterile filtration membranes. Generally, dispersions are prepared
by incorporating the various sterilized active ingredients into a
sterile vehicle which contains the basic dispersion medium and/or
the other ingredients. In the case of sterile powders for the
preparation of sterile injectable solutions, suspensions or
emulsion, the preferred methods of preparation are vacuum-drying or
freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered liquid medium thereof. The liquid medium should be
suitably buffered if necessary and the liquid diluent first
rendered isotonic prior to injection with sufficient saline or
glucose. The composition must be stable under the conditions of
manufacture and storage, and preserved against the contaminating
action of microorganisms, such as bacteria and fungi. It will be
appreciated that endotoxin contamination should be kept minimally
at a safe level, for example, less than 0.5 ng/mg protein. Suitable
pharmaceutically acceptable excipients include, but are not limited
to: 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
polyethylene glycol (PEG). Aqueous injection suspensions may
contain compounds which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, dextran, or the
like. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl cleats or
triglycerides, or liposomes.
[0601] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing
Company, 1990). Sustained-release preparations may be prepared.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
polypeptide, which matrices are in the form of shaped articles,
e.g. films, or microcapsules. In particular embodiments, prolonged
absorption of an injectable composition can be brought about by the
use in the compositions of agents delaying absorption, such as, for
example, aluminum monostearate, gelatin or combinations
thereof.
[0602] Exemplary pharmaceutically acceptable excipients herein
further include insterstitial drug dispersion agents such as
soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for
example, human soluble PH-20 hyaluronidase glycoproteins, such as
rHuPH20 (HYLENEX.RTM., Baxter International, Inc.). Certain
exemplary sHASEGPs and methods of use, including rHuPH20, are
described in US Patent Publication Nos. 2005/0260186 and
2006/0104968. In one aspect, a sHASEGP is combined with one or more
additional glycosaminoglycanases such as chondroitinases.
[0603] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the
latter formulations including a histidine-acetate buffer.
[0604] In addition to the compositions described previously, the
antigen binding molecules may also be formulated as a depot
preparation. Such long acting formulations may be administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection. Thus, for example, the fusion proteins may
be formulated with suitable polymeric or hydrophobic materials (for
example as emulsion in an acceptable oil) or ion exchange resins,
or as sparingly soluble derivatives, for example, as a sparingly
soluble salt.
[0605] Pharmaceutical compositions comprising the bispecific
antigen binding molecules of the invention may be manufactured by
means of conventional mixing, dissolving, emulsifying,
encapsulating, entrapping or lyophilizing processes. Pharmaceutical
compositions may be formulated in conventional manner using one or
more physiologically acceptable carriers, diluents, excipients or
auxiliaries which facilitate processing of the proteins into
preparations that can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen.
[0606] The bispecific antigen binding molecules may be formulated
into a composition in a free acid or base, neutral or salt form.
Pharmaceutically acceptable salts are salts that substantially
retain the biological activity of the free acid or base. These
include the acid addition salts, e.g. those formed with the free
amino groups of a proteinaceous composition, or which are formed
with inorganic acids such as for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic, tartaric
or mandelic acid. Salts formed with the free carboxyl groups can
also be derived from inorganic bases such as for example, sodium,
potassium, ammonium, calcium or ferric hydroxides; or such organic
bases as isopropylamine, trimethylamine, histidine or procaine.
Pharmaceutical salts tend to be more soluble in aqueous and other
protic solvents than are the corresponding free base forms.
[0607] The composition herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended.
[0608] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0609] Therapeutic Methods and Compositions
[0610] Any of the bispecific antigen binding molecules provided
herein may be used in therapeutic methods. For use in therapeutic
methods, bispecific antigen binding molecules of the invention can
be formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical
practitioners.
[0611] In one aspect, bispecific antigen binding molecules of the
invention for use as a medicament are provided.
[0612] In further aspects, bispecific antigen binding molecules of
the invention for use (i) in inducing immune stimulation by
CD40.sup.+ antigen-presenting cells (APCs), (ii) in stimulating
tumor-specific T cell response, (iii) in causing apoptosis of tumor
cells, (iv) in the treatment of cancer, (v) in delaying progression
of cancer, (vi) in prolonging the survival of a patient suffering
from cancer, (vii) in the treatment of infections are provided. In
a particular aspect, bispecific antigen binding molecules of the
invention for use in treating a disease, in particular for use in
the treatment of cancer, are provided.
[0613] In certain aspects, bispecific antigen binding molecules of
the invention for use in a method of treatment are provided. In one
aspect, the invention provides a bispecific antigen binding
molecule as described herein for use in the treatment of a disease
in an individual in need thereof. In certain aspects, the invention
provides a bispecific antigen binding molecule for use in a method
of treating an individual having a disease comprising administering
to the individual a therapeutically effective amount of the
bispecific antigen binding molecule. In certain aspects the disease
to be treated is cancer. The subject, patient, or "individual" in
need of treatment is typically a mammal, more specifically a
human.
[0614] In one aspect, provided is a method for i) inducing immune
stimulation by CD40+ antigen-presenting cells (APCs), (ii)
stimulating tumor-specific T cell response, (iii) causing apoptosis
of tumor cells, (iv) treating of cancer, (v) delaying progression
of cancer, (vi) prolonging the survival of a patient suffering from
cancer, or (vii) treating of infections, wherein the method
comprises administering a therapeutically effective amount of the
bispecific antigen binding molecule of the invention to an
individual in need thereof.
[0615] In a further aspect, the invention provides for the use of
the bispecific antigen binding molecule of the invention in the
manufacture or preparation of a medicament for the treatment of a
disease in an individual in need thereof. In one aspect, the
medicament is for use in a method of treating a disease comprising
administering to an individual having the disease a therapeutically
effective amount of the medicament. In certain aspects, the disease
to be treated is a proliferative disorder, particularly cancer.
Examples of cancers include, but are not limited to, bladder
cancer, brain cancer, head and neck cancer, pancreatic cancer, lung
cancer, breast cancer, ovarian cancer, uterine cancer, cervical
cancer, endometrial cancer, esophageal cancer, colon cancer,
colorectal cancer, rectal cancer, gastric cancer, prostate cancer,
blood cancer, skin cancer, squamous cell carcinoma, bone cancer,
and kidney cancer. Other examples of cancer include carcinoma,
lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma,
sarcoma, and leukemia. Other cell proliferation disorders that can
be treated using the bispecific antigen binding molecule or
antibody of the invention include, but are not limited to neoplasms
located in the: abdomen, bone, breast, digestive system, liver,
pancreas, peritoneum, endocrine glands (adrenal, parathyroid,
pituitary, testicles, ovary, thymus, thyroid), eye, head and neck,
nervous system (central and peripheral), lymphatic system, pelvic,
skin, soft tissue, spleen, thoracic region, and urogenital system.
Also included are pre-cancerous conditions or lesions and cancer
metastases. In certain embodiments the cancer is chosen from the
group consisting of renal cell cancer, skin cancer, lung cancer,
colorectal cancer, breast cancer, brain cancer, head and neck
cancer. A skilled artisan readily recognizes that in many cases the
the bispecific antigen binding molecule or antibody of the
invention may not provide a cure but may provide a benefit. In some
aspects, a physiological change having some benefit is also
considered therapeutically beneficial. Thus, in some aspects, an
amount of the bispecific antigen binding molecule or antibody of
the invention that provides a physiological change is considered an
"effective amount" or a "therapeutically effective amount".
[0616] For the prevention or treatment of disease, the appropriate
dosage of a bispecific antigen binding molecule of the invention
(when used alone or in combination with one or more other
additional therapeutic agents) will depend on the type of disease
to be treated, the route of administration, the body weight of the
patient, the specific molecule, the severity and course of the
disease, whether the bispecific antigen binding molecule of the
invention is administered for preventive or therapeutic purposes,
previous or concurrent therapeutic interventions, the patient's
clinical history and response to the bispecific antigen binding
molecule, and the discretion of the attending physician. The
practitioner responsible for administration will, in any event,
determine the concentration of active ingredient(s) in a
composition and appropriate dose(s) for the individual subject.
Various dosing schedules including but not limited to single or
multiple administrations over various time-points, bolus
administration, and pulse infusion are contemplated herein.
[0617] The bispecific antigen binding molecule of the invention is
suitably administered to the patient at one time or over a series
of treatments. Depending on the type and severity of the disease,
about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of the
bispecific antigen binding molecule can be an initial candidate
dosage for administration to the patient, whether, for example, by
one or more separate administrations, or by continuous infusion.
One typical daily dosage might range from about 1 .mu.g/kg to 100
mg/kg or more, depending on the factors mentioned above. For
repeated administrations over several days or longer, depending on
the condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the bispecific antigen binding molecule of the invention
would be in the range from about 0.005 mg/kg to about 10 mg/kg. In
other examples, a dose may also comprise from about 1 .mu.g/kg body
weight, about 5 .mu.g/kg body weight, about 10 .mu.g/kg body
weight, about 50 .mu.g/kg body weight, about 100 .mu.g/kg body
weight, about 200 .mu.g/kg body weight, about 350 .mu.g/kg body
weight, about 500 .mu.g/kg body weight, about 1 mg/kg body weight,
about 5 mg/kg body weight, about 10 mg/kg body weight, about 50
mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg
body weight, about 350 mg/kg body weight, about 500 mg/kg body
weight, to about 1000 mg/kg body weight or more per administration,
and any range derivable therein. In examples of a derivable range
from the numbers listed herein, a range of about 0.1 mg/kg body
weight to about 20 mg/kg body weight, about 5 .mu.g/kg body weight
to about 1 mg/kg body weight etc., can be administered, based on
the numbers described above. Thus, one or more doses of about 0.5
mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination
thereof) may be administered to the patient. Such doses may be
administered intermittently, e.g. every week or every three weeks
(e.g. such that the patient receives from about two to about
twenty, or e.g. about six doses of the fusion protein). In a
particular aspect, the bispecific antigen binding molecule will be
administered every three weeks. An initial higher loading dose,
followed by one or more lower doses may be administered. However,
other dosage regimens may be useful. The progress of this therapy
is easily monitored by conventional techniques and assays.
[0618] The bispecific antigen binding molecule of the invention
will generally be used in an amount effective to achieve the
intended purpose. For use to treat or prevent a disease condition,
the bispecific antigen binding molecule of the invention, or
pharmaceutical compositions thereof, are administered or applied in
a therapeutically effective amount. Determination of a
therapeutically effective amount is well within the capabilities of
those skilled in the art, especially in light of the detailed
disclosure provided herein. For systemic administration, a
therapeutically effective dose can be estimated initially from in
vitro assays, such as cell culture assays. A dose can then be
formulated in animal models to achieve a circulating concentration
range that includes the IC.sub.50 as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Initial dosages can also be estimated from in vivo
data, e.g., animal models, using techniques that are well known in
the art. One having ordinary skill in the art could readily
optimize administration to humans based on animal data.
[0619] Dosage amount and interval may be adjusted individually to
provide plasma levels of the bispecific antigen binding molecule of
the invention which are sufficient to maintain therapeutic effect.
Usual patient dosages for administration by injection range from
about 0.1 to 50 mg/kg/day, typically from about 0.1 to 1 mg/kg/day.
Therapeutically effective plasma levels may be achieved by
administering multiple doses each day. Levels in plasma may be
measured, for example, by HPLC. In cases of local administration or
selective uptake, the effective local concentration of the
bispecific antigen binding molecule or antibody of the invention
may not be related to plasma concentration. One skilled in the art
will be able to optimize therapeutically effective local dosages
without undue experimentation.
[0620] A therapeutically effective dose of the bispecific antigen
binding molecule of the invention described herein will generally
provide therapeutic benefit without causing substantial toxicity.
Toxicity and therapeutic efficacy of a fusion protein can be
determined by standard pharmaceutical procedures in cell culture or
experimental animals. Cell culture assays and animal studies can be
used to determine the LD.sub.50 (the dose lethal to 50% of a
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of a population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, which can be
expressed as the ratio LD.sub.50/ED.sub.50. Bispecific antigen
binding molecules that exhibit large therapeutic indices are
preferred. In one aspect, the the bispecific antigen binding
molecule or antibody of the invention exhibits a high therapeutic
index. The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosages suitable for
use in humans. The dosage lies preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage may vary within this range depending upon a
variety of factors, e.g., the dosage form employed, the route of
administration utilized, the condition of the subject, and the
like. The exact formulation, route of administration and dosage can
be chosen by the individual physician in view of the patient's
condition (see, e.g., Fingl et al., 1975, in: The Pharmacological
Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by
reference in its entirety).
[0621] The attending physician for patients treated with fusion
proteins of the invention would know how and when to terminate,
interrupt, or adjust administration due to toxicity, organ
dysfunction, and the like. Conversely, the attending physician
would also know to adjust treatment to higher levels if the
clinical response were not adequate (precluding toxicity). The
magnitude of an administered dose in the management of the disorder
of interest will vary with the severity of the condition to be
treated, with the route of administration, and the like. The
severity of the condition may, for example, be evaluated, in part,
by standard prognostic evaluation methods. Further, the dose and
perhaps dose frequency will also vary according to the age, body
weight, and response of the individual patient.
[0622] Other Agents and Treatments
[0623] The bispecific antigen binding molecule of the invention may
be administered in combination with one or more other agents in
therapy. For instance, the bispecific antigen binding molecule or
antibody of the invention of the invention may be co-administered
with at least one additional therapeutic agent. The term
"therapeutic agent" encompasses any agent that can be administered
for treating a symptom or disease in an individual in need of such
treatment. Such additional therapeutic agent may comprise any
active ingredients suitable for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. In certain embodiments, an additional
therapeutic agent is another anti-cancer agent, for example a
microtubule disruptor, an antimetabolite, a topoisomerase
inhibitor, a DNA intercalator, an alkylating agent, a hormonal
therapy, a kinase inhibitor, a receptor antagonist, an activator of
tumor cell apoptosis, or an antiangiogenic agent. In certain
aspects, an additional therapeutic agent is an immunomodulatory
agent, a cytostatic agent, an inhibitor of cell adhesion, a
cytotoxic or cytostatic agent, an activator of cell apoptosis, or
an agent that increases the sensitivity of cells to apoptotic
inducers.
[0624] Thus, provided are bispecific antigen binding molecules of
the invention or pharmaceutical compositions comprising them for
use in the treatment of cancer, wherein the bispecific antigen
binding molecule is administered in combination with a
chemotherapeutic agent, radiation and/or other agents for use in
cancer immunotherapy.
[0625] Such other agents are suitably present in combination in
amounts that are effective for the purpose intended. The effective
amount of such other agents depends on the amount of fusion protein
used, the type of disorder or treatment, and other factors
discussed above. The the bispecific antigen binding molecule or
antibody of the invention are generally used in the same dosages
and with administration routes as described herein, or about from 1
to 99% of the dosages described herein, or in any dosage and by any
route that is empirically/clinically determined to be
appropriate.
[0626] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate compositions), and separate administration,
in which case, administration of the bispecific antigen binding
molecule or antibody of the invention can occur prior to,
simultaneously, and/or following, administration of the additional
therapeutic agent and/or adjuvant.
[0627] Articles of Manufacture
[0628] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper that is
pierceable by a hypodermic injection needle). At least one active
agent in the composition is a bispecific antigen binding molecule
of the invention.
[0629] The label or package insert indicates that the composition
is used for treating the condition of choice. Moreover, the article
of manufacture may comprise (a) a first container with a
composition contained therein, wherein the composition comprises a
bispecific antigen binding molecule of the invention; and (b) a
second container with a composition contained therein, wherein the
composition comprises a further cytotoxic or otherwise therapeutic
agent. The article of manufacture in this embodiment of the
invention may further comprise a package insert indicating that the
compositions can be used to treat a particular condition.
[0630] Alternatively, or additionally, the article of manufacture
may further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
TABLE-US-00003 TABLE C (Sequences): SEQ ID NO: Name Sequence 1 hu
CD40 UniProt no. P25942, version 200 MVRLPLQCVL WGCLLTAVHP
EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQH
KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD
TICEPCPVGF FSNVSSAFEK CHPWTSCETK DLVVQQAGTN KTDVVCGPQD RLRALVVIPI
IFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP
VTQEDGKESR ISVQERQ 2 hu FAP UniProt no. Q12884, version 168
MKTWVKIVFG VATSAVLALL VMCIVLRPSR VHNSEENTMR ALTLKDILNG TFSYKTFFPN
WISGQEYLHQ SADNNIVLYN IETGQSYTIL SNRTMKSVNA SNYGLSPDRQ FVYLESDYSK
LWRYSYTATY YIYDLSNGEF VRGNELPRPI QYLCWSPVGS KLAYVYQNNI YLKQRPGDPP
FQITFNGREN KIFNGIPDWV YEEEMLATKY ALWWSPNGKF LAYAEFNDTD IPVIAYSYYG
DEQYPRTINI PYPKAGAKNP VVRIFIIDTT YPAYVGPQEV PVPAMIASSD YYFSWLTWVT
DERVCLQWLK RVQNVSVLSI CDFREDWQTW DCPKTQEHIE ESRTGWAGGF FVSTPVFSYD
AISYYKIFSD KDGYKHIHYI KDTVENAIQI TSGKWEAINI FRVTQDSLFY SSNEFEEYPG
RRNIYRISIG SYPPSKKCVT CHLRKERCQY YTASFSDYAK YYALVCYGPG IPISTLHDGR
TDQEIKILEE NKELENALKN IQLPKEEIKK LEVDEITLWY KMILPPQFDR SKKYPLLIQV
YGGPCSQSVR SVFAVNWISY LASKEGMVIA LVDGRGTAFQ GDKLLYAVYR KLGVYEVEDQ
ITAVRKFIEM GFIDEKRIAI WGWSYGGYVS SLALASGTGL FKCGIAVAPV SSWEYYASVY
TERFMGLPTK DDNLEHYKNS TVMARAEYFR NVDYLLIHGT ADDNVHFQNS AQIAKALVNA
QVDFQAMWYS DQNHGLSGLS TNHLYTHMTH FLKQCFSLSD 3 FAP (28H1) CDR-H1
SHAMS 4 FAP (28H1) CDR-H2 AIWASGEQYYADSVKG 5 FAP (28H1) CDR-H3
GWLGNFDY 6 FAP (28H1) CDR-L1 RASQSVSRSYLA 7 FAP (28H1) CDR-L2
GASTRAT 8 FAP (28H1) CDR-L3 QQGQVIPPT 9 FAP(28H1) VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMS
WVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTIS
RDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDY WGQGTLVTVSS 10 FAP(28H1) VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLA
WYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGT
DFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKV EIK 11 FAP(4B9) CDR-H1 SYAMS 12
FAP(4B9) CDR-H2 AIIGSGASTYYADSVKG 13 FAP(4B9) CDR-H3 GWFGGFNY 14
FAP(4B9) CDR-L1 RASQSVTSSYLA 15 FAP(4B9) CDR-L2 VGSRRAT 16 FAP(4B9)
CDR-L3 QQGIMLPPT 17 FAP(4B9) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMS
WVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTI
SRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFN YWGQGTLVTVSS 18 FAP(4B9) VL
EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLA
WYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGT
DFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKV EIK 19 hu CD40 CDR-H1 GYYIH 20
hu CD40 CDR-H2 RVIPNAGGTSYNQKFKG 21 hu CD40 CDR-H3 EGIYW 22 hu CD40
CDR-L1 RSSQSLVHSNGNTFLH 23 hu CD40 CDR-L2 TVSNRFS 24 hu CD40 CDR-L3
SQTTHVPWT 25 hu CD40 VH EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIH
WVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTL
SVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWG QGTLVTVSS 26 hu CD40 VL
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGN
TFLHWYQQKPGKAPKLLIYTVSNRFSGVPSRFSGS
GSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQ GTKVEIK 27 mu CD40 CDR-H1 DYYMA
28 mu CD40 CDR-H2 SISYDGSSTYYRDSVKG 29 mu CD40 CDR-H3 HSSYFDY 30 mu
CD40 CDR-L1 ASDSVSTLMH 31 mu CD40 CDR-L2 LASHLES 32 mu CD40 CDR-L3
QQSWNDPWT 33 mu CD40 VH EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMA
WVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTI
SRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDY WGQGVMVTVSS 34 mu CD40 VL
DTVLTQSPALAVSPGERVTISCRASDSVSTLMHWY
QQKPGQQPKLLIYLASHLESGVPARFSGSGSGTDF
TLTIDPVEADDTATYYCQQSWNDPWTFGGGTKLEL K 35 hu CD40 CDR-H1 long
GYSFTGYYIH 36 hu CD40 CDR-H2 (hVH_1, RVIPNNGGTSYNQKFKG hVH_2) 37 hu
CD40 CDR-H2 (hVH_3) RVIPNAGGTSYNQKFKG 38 hu CD40 CDR-H2 (hVH_4)
RVIPQAGGTSYNQKFKG 39 hu CD40 CDR-H2 (hVH_5) RVIPNNGGTSYNQKFQG 40 hu
CD40 CDR-H2 (hVH_6) RVIPNNGGTSYAQKFKG 41 hu CD40 CDR-H2 (hVH_7)
RVIPNNGGTSYAQKFQG 42 hu CD40 CDR-H2 RVIPNAGGTSYNQKFQG (hVH_5_N288A)
43 hu CD40 CDR-H2 RVIPNAGGTSYAQKFKG (hVH_6_N288A) 44 hu CD40 CDR-H2
RVIPNAGGTSYAQKFQG (hVH_7_N288A) 45 hu CD40 hVH_1 see Table 14 46 hu
CD40 hVH_2 see Table 14 47 hu CD40 hVH_3 see Table 14 48 hu CD40
hVH_4 see Table 14 49 hu CD40 hVH_5 see Table 14 50 hu CD40 hVH_6
see Table 14 51 hu CD40 hVH_7 see Table 14 52 hu CD40 hVH_2_N288A
see Table 14 53 hu CD40 hVH_5_N288A see Table 14 54 hu CD40
hVH_6_N288A see Table 14 55 hu CD40 hVH_7_N288A see Table 14 56 hu
CD40 hVK_1 see Table 15 57 hu CD40 hVK_2 see Table 15 58 hu CD40
hVK_3 see Table 15 59 hu CD40 hVK_4 see Table 15 60 hu CD40 hVK_5
see Table 15 61 hu CD40 hVK_6 see Table 15 62 hu CD40 hVK_7 see
Table 15 63 hu CD40 hVK_8 see Table 15 64 hu CD40 hVK_9 see Table
15 65 DP47-CDR H1 SYAMS 66 DP47-CDR H2 AISGSGGSTYYADSVKG 67
DP47-CDR H3 GSGFDY 68 DP47-CDR L1 RASQSVSSSYLA 69 DP47-CDR L2
GASSRAT 70 DP47-CDR L3 QQYGSSPLT 71 DP47 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMS
WVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI
SRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYW GQGTLVTVSS 72 DP47 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLA
WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT
DFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKV EIK 73 hu CD40 VH (nucleotide
see Table 2 sequence) 74 hu CD40 VL (nucleotide see Table 2
sequence) 75 FAP 28H1 VH (nucleotide see Table 2 sequence) 76 FAP
28H1 VL (nucleotide see Table 2 sequence) 77 DP47 VH (nucleotide
see Table 2 sequence) 78 DP47 VL(nucleotide see Table 2 sequence)
79 mu CD40 VH (nucleotide see Table 2 sequence) 80 mu CD40 VL
(nucleotide see Table 2 sequence) 81 CD40 IgG heavy chain see Table
3 82 CD40 light chain see Table 3 83 CD40 VHCH1-CD40 see Table 3
VHCH1-Fcknob_PGLALA-
28H1 VH 84 CD40 VHCH1-CD40 see Table 3 VHCH1-Fchole_PGLALA- 28H1 VL
85 CD40 VHCH1-CD40 see Table 3 VHCH1-Fc_PGLALA- 28H1 VLCH1 EE 86
CD40 light chain RK see Table 3 87 28H1 VHCL see Table 3 88
huCD40_Fchole_PGLALA_28H1 see Table 3 VL 89
huCD40_Fcknob_PGLALA_28H1 see Table 3 VH 90 huCD40- see Table 3
Fc_PGLALA_28H1_VLCH1 EE 91 CD40 VHCH1-CD40 see Table 3
VHCH1-Fcknob_PGLALA- DP47 VH 92 CD40 VHCH1-CD40 see Table 3
VHCH1-Fchole_PGLALA- DP47 VL 93 CD40 VHCH1-CD40 see Table 3
VHCH1-Fc_PGLALA- DP47 VLCH1 EE 94 DP47VHCL see Table 3 95 muCD40
VHCH1-muCD40 see Table 3 VHCH1-FcKK_DAPG- 28H1 VH 96 muCD40
VHCH1-muCD40 see Table 3 VHCH1-FcDD_DAPG- 28H1 VL 97 mu CD40 light
chain see Table 3 98 muCD40 VHCH1-muCD40 see Table 3
VHCH1-Fc_DAPG-28H1 VLCH1 99 28H1 VHCL (mu) see Table 3 100 mu CD40
light chain; `RK` see Table 3 101 muCD40 VHCH1-muCD40 see Table 3
VHCH1-FcKK_DAPG- DP47 VH 102 muCD40 VHCH1-mu CD40 see Table 3
VHCH1-FcDD_DAPG- DP47 VL 103 muCD40 VHCH1-muCD40 see Table 3
VHCH1-Fc_DAPG-28H1 VLCH1 104 DP47 VHCL (mu) see Table 3 105 CD40
IgG heavy chain see Table 4 (nucleotide sequence) 106 CD40 light
chain (nucleotide see Table 4 sequence) 107 CD40 VHCH1-CD40 see
Table 4 VHCH1-Fcknob_PGLALA- 28H1 VH 108 CD40 VHCH1-CD40 see Table
4 VHCH1-Fchole_PGLALA- 28H1 VL 109 CD40 VHCH1-CD40 see Table 4
VHCH1-Fc_PGLALA- 28H1 VLCH1 EE 110 CD40 light chain RK see Table 4
111 28H1 VHCL see Table 4 112 huCD40_Fchole_PGLALA_28H1 see Table 4
VL 113 huCD40_Fcknob_PGLALA_28H1 see Table 4 VH 114 huCD40- see
Table 4 Fc_PGLALA_28H1_VLCH1 EE 115 CD40 VHCH1-CD40 see Table 4
VHCH1-Fcknob_PGLALA- DP47 VH 116 CD40 VHCH1-CD40 see Table 4
VHCH1-Fchole_PGLALA- DP47 VL 117 CD40 VHCH1-CD40 see Table 4
VHCH1-Fc_PGLALA- DP47 VLCH1 EE 118 DP47 VHCL see Table 4 119 muCD40
VHCH1-muCD40 see Table 4 VHCH1-FcKK_DAPG- 28H1 VH 120 muCD40
VHCH1-muCD40 see Table 4 VHCH1-FcDD_DAPG- 28H1 VL 121 mu CD40 light
chain see Table 4 122 muCD40 VHCH1-muCD40 see Table 4
VHCH1-Fc_DAPG-28H1 VLCH1 123 28H1 VHCL (mu) see Table 4 124 mu CD40
light chain; `RK` see Table 4 125 muCD40 VHCH1-muCD40 see Table 4
VHCH1-FcKK_DAPG- DP47 VH 126 muCD40 VHCH1-mu CD40 see Table 4
VHCH1-FcDD_DAPG- DP47 VL 127 muCD40 VHCH1-muCD40 see Table 4
VHCH1-Fc_DAPG-28H1 VLCH1 128 DP47 VHCL (mu) see Table 4 129 CD40
(S2C6) VH EVQLQQSGPD LVKPGASVKI SCKASGYSFT GYYIHWVKQS HGKSLEWIGR
VIPNNGGTSY NQKFKGKAIL TVDKSSSTAY MELRSLTSED SAVYYCAREG IYWWGHGTTL
TVSS 130 CD40 (S2C6) VL DVVVTQTPLS LPVSLGAQAS ISCRSSQSLV HSNGNTFLHW
YLQKPGQSPK LLIYTVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQTTHVP
WTFGGGTKLE IQ 131 hVH3_CD40 VHCH1- see Table 27 VHCH1-Fc
knob_PGLALA- 4B9 VH 132 hVH3_CD40 VHCH1- see Table 27 VHCH1-Fc
hole_PGLALA- 4B9 VL 133 hVK2_CD40 light chain see Table 27 134
hVH3_CD40 VHCH1-Fcknob_PGLALA- see Table 27 4B9 VH 135 hVH3_CD40
VHCH1-Fchole_PGLALA- see Table 27 4B9 VL 136 hVH3_CD40-Fc
knob_PGLALA_4B9_VLCH1 see Table 27 `EE` 137 hVK2_CD40 LC ,RK` see
Table 27 138 4B9 VHCL see Table 27 139 hVH3_CD40-Fchole_PGLALA see
Table 27 `EE` 140 hVH3_CD40-Fchole_PGLALA see Table 27 `EE` 141
4B9-Fc knob_PGLALA see Table 27 142 hu FAP ectodomain + poly-
RPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNW lys-tag + his.sub.6-tag
ISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMK
SVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYY
IYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVY
QNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVY
EEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAY
SYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTY
PAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCL
QWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEE
SRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKH
IHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYS
SNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKE
RCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRT
DQEIKILEENKELENALKNIQLPKEEIKKLEVDEI
TLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRS
VFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLY
AVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIW
GWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYY
ASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRN
VDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQA
MWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDG KKKKKKGHHHHHH 143 mouse FAP
UniProt no. P97321 144 Murine FAP
RPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNW ectodomain + poly-lys-
ISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMK tag + his.sub.6-tag
SVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYY
IYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVY
QNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVY
EEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAY
SYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTY
PHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCL
QWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEE
SRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKH
IHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYS
SNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKE
RCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRT
DQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGL
TFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKS
VFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLH
AVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIW
GWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYY
ASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRN
VDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQA
MWYSDQNHGILSGRSQNHLYTHMTHFLKQCFSLSD GKKKKKKGHHHHHH 145 Cynomolgus
FAP RPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNW ectodomain + poly-lys-
ISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMK tag + his.sub.6-tag
SVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYY
IYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVY
QNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVY
EEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAY
SYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTY
PAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCL
QWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEE
SRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKH
IHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYS
SNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKE
RCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRT
DQEIKILEENKELENALKNIQLPKEEIKKLEVDEI
TLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRS
VFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLY
AVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIW
GWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYY
ASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRN
VDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQA
MWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDG KKKKKKGHHHHHH 146 murine CD40
UniProt P27512, version 160 MVSLPRLCAL WGCLLTAVHL GQCVTCSDKQ
YLHDGQCCDL CQPGSRLTSH CTALEKTQCH PCDSGEFSAQ WNREIRCHQH RHCEPNQGLR
VKKEGTAESD TVCTCKEGQH CTSKDCEACA QHTPCIPGFG VMEMATETTD TVCHPCPVGF
FSNQSSLFEK CYPWTSCEDK NLEVLQKGTS QTNVICGLKS RMRALLVIPV VMGILITIFG
VFLYIKKVVK KPKDNEILPP AARRQDPQEM EDYPGHNTAA PVQETLHGCQ PVTQEDGKES
RISVQERQVT DSIALRPLV 147 Peptide linker (G4S) GGGGS 148 Peptide
linker (G4S).sub.2 GGGGSGGGGS 149 Peptide linker (SG4).sub.2
SGGGGSGGGG 150 Peptide linker G4(SG4).sub.2 GGGGSGGGGSGGGG 151
peptide linker GSPGSSSSGS 152 (G4S).sub.3 peptide linker
GGGGSGGGGSGGGGS.sub.3 153 (G4S).sub.4 peptide linker
GGGGSGGGGSGGGGSGGGGS 154 peptide linker GSGSGSGS 155 peptide linker
GSGSGNGS 156 peptide linker GGSGSGSG 157 peptide linker GGSGSG 158
peptide linker GGSG 159 peptide linker GGSGNGSG 160 peptide linker
GGNGSGSG 161 peptide linker GGNGSG 162 28H1 light chain cross see
Table 6 VHCL 163 28H1 (VLCH1)_Fc knob_PGLALA see Table 6 164 CD40
(VHCH1 charged)_Fchole_PGLALA see Table 6 165 CD40 light chain
(charged) see Table 6 166 CD40 (VHCH1 see Table 6 charged)_28H1
(VLCH1)_FC knob_PGLALA 167 CD40 (VHCH1 charged)_Fcknob_PGLALA_28H1
see Table 6 (VLCH1) 168 CD40 (VHCH1 charged)_Fchole_PGLALA see
Table 6 169 CD40 (VHCH1 see Table 6 charged_CD40 (VHCH1
charged)-Fcknob_PGLALA_28H1 (VLCH1) 170 CD40 (VHCH1 see Table 6
charged_CD40 (VHCH1 charged)-Fc hole_PGLALA 171 VH1a (CD40) see
Table 20 172 VH1b (CD40) see Table 20 173 VH1c (CD40) see Table 20
174 VH1d (CD40) see Table 20 175 VL1a (CD40) see Table 20 176 VL1b
(CD40) see Table 20 177 VL1c (CD40) see Table 20 178 VL1d (CD40)
see Table 20 179 VH2a (CD40) see Table 21 180 VH2b (CD40) see Table
21 181 VH2c (CD40) see Table 21 182 VH2d (CD40) see Table 21 183
VH2ab (CD40) see Table 21 184 VH2ac (CD40) see Table 21 185 VL2a
(CD40) see Table 21 186 VL2b (CD40) see Table 21 187 VL2ab (CD40)
see Table 21 188 VL2ac (CD40) see Table 21 189 P1AE0400 Heavy chain
see Table 23 190 P1AE0400 light chain see Table 23 191 P1AE0401
heavy chain see Table 23 192 P1AE0401 light chain see Table 23 193
P1AE0402 heavy chain see Table 23 194 P1AE0402 light chain see
Table 23 195 P1AE0403 heavy chain see Table 23 196 P1AE0403 light
chain see Table 23 197 P1AE0404 heavy chain see Table 23 198
P1AE0404 light chain see Table 23 199 P1AE0405 heavy chain see
Table 23 200 P1AE0405 light chain see Table 23 201 P1AE0406 heavy
chain see Table 23 202 P1AE0406 light chain see Table 23 203
P1AE0407 heavy chain see Table 23 204 P1AE0407 light chain see
Table 23 205 P1AE0817 heavy chain see Table 23 206 P1AE0817 light
chain see Table 23 207 P1AE0818 heavy chain see Table 23 208
P1AE0818 light chain see Table 23 209 P1AE0819 heavy chain see
Table 23 210 P1AE0819 light chain see Table 23 211 P1AE0993 heavy
chain see Table 23 212 P1AE0993 light chain see Table 23 213
P1AE0996 heavy chain see Table 23 214 P1AE0996 light chain see
Table 23 215 P1AE0997 heavy chain see Table 23 216 P1AE0997 light
chain see Table 23 217 P1AE0998 heavy chain see Table 23 218
P1AE0998 light chain see Table 23 219 P1AE0999 heavy chain see
Table 23 220 P1AE0999 light chain see Table 23 221 P1AE1000 heavy
chain see Table 23 222 P1AE1000 light chain see Table 23 223
P1AE1001 heavy chain see Table 23 224 P1AE1001 light chain see
Table 23 225 P1AE1002 heavy chain see Table 23 226 P1AE1002 light
chain see Table 23 227 P1AE1003 heavy chain see Table 23 228
P1AE1003 light chain see Table 23 229 P1AE1004 heavy chain see
Table 23 230 P1AE1004 light chain see Table 23 231 P1AE1005 heavy
chain see Table 23 232 P1AE1005 light chain see Table 23 233
P1AE1006 heavy chain see Table 23 234 P1AE1006 light chain see
Table 23 235 P1AE1007 heavy chain see Table 23 236 P1AE1007 light
chain see Table 23 237 P1AE1125 heavy chain see Table 23 238
P1AE1125 light chain see Table 23 239 P1AE1126 heavy chain see
Table 23 240 P1AE1126 light chain see Table 23 241 P1AE1135 heavy
chain see Table 23 242 P1AE1135 light chain see Table 23 243 VL2a
(CD40) light chain see Table 28 (charged) 244 VH2a (CD40) (VHCH1
see Table 28 charged_VH2a (CD40) (VHCH1 charged)-Fcknob_PGLALA_28H1
(VLCH1) 245 VH2a (CD40) (VHCH1 see Table 28 charged_VH2a (CD40)
(VHCH1 charged)-Fchole_PGLALA 246 VH2d (CD40) (VHCH1 see Table 28
charged_VH2d (CD40) (VHCH1 charged)-Fcknob_PGLALA_28H1 (VLCH1) 247
VH2d (CD40) (VHCH1 see Table 28 charged_VH2d (CD40) (VHCH1
charged)-Fchole_PGLALA 248 VL1a (CD40) light chain see Table 28
(charged) 249 VH1a (CD40) (VHCH1)_VH1a see Table 28 (CD40) (VHCH1)
Fcknob_PGLALA_28H1 (VLCH1) (charged)
250 VH1a (CD40) (VHCH1)_VH1a see Table 28 (CD40) (VHCH1)- Fc
hole_PGLALA (charged) 251 VH1a (CD40) (VHCH1) Fcknob_PGLALA_28H1
see Table 28 (VLCH1) (charged) 252 VH1a (CD40) (VHCH1)
Fchole_PGLALA see Table 28 (charged) 253 VH1a (CD40) (VHCH1)
Fcknob_PGLALA_4B9 see Table 28 (VLCH1) (charged) 254 4B9 light
chain cross VLCH see Table 28 255 VH1a (CD40) (VHCH1)
Fcknob_PGLALA_4B9 see Table 28 (VHCL) (charged) 256 VL1a (CD40)
light chain see Table 28 257 VH1a (CD40) (VHCH1) Fcknob_PGLALA_4B9
see Table 28 (VHCL) 258 VH1a (CD40) (VHCH1) Fchole_PGLALA see Table
28 259 P1AE0816 heavy chain see Table 23 (control) 260 P1AE0816
light chain see Table 23 (control) 261 hu CD40 CDR-H1 (VH2ab) GYYMH
262 hu CD40 CDR-H2 (VH2ab) RVIPNAGGTSYNQKFKG 263 hu CD40 CDR-H2
(VH2ac) RVIPNAGGTSYNQKVKG 264 hu CD40 CDR-L1 (VL2ab)
RASQSLVHSNGNTFLH 265 hu CD40 CDR-L1 (VL2ac) RSSQSIVHSNGNTFLH 266
Hu_CD40_ECD_His_Avi EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFT
ETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLR
VQQKGTSETDTICTCEEGWHCTSEACESCVLHRSC
SPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEK
CHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRGG GGSHHHHHHGSGLNDIFEAQKIEWHE 267
cyno_CD40_ECD_His_Avi EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFT
ETECLPCSESEFLDTWNRETRCHQHKYCDPNLGLR
VQQKGTSETDTICTCEEGLHCTSESCESCVPHRSC
LPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEK
CRPWTSCETKDLVVQQAGTNKTDVVCGPQDRQRGG GGSHHHHHHGSGLNDIFEAQKIEWHE 268
Selicrelumab IgG2 heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMH chain
WVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTM (control)
TRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYC
TNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPC
SRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCN
VDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
NWYVDGVEVHNAKTKPREEQFNSTRFVVSVLTVVH
QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
269 Selicrelumab IgG2 light DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAW
chain YQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTD (control)
FTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVE
IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY
PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC
[0631] The following numbered paragraphs (paras) describe aspects
of the present invention according to the first priority
application:
[0632] 1. A bispecific antigen binding molecule, comprising [0633]
(a) at least one antigen binding domain capable of specific binding
to CD40, and [0634] (b) at least one antigen binding domain capable
of specific binding to a target cell antigen.
[0635] 2. The bispecific antigen binding molecule of para 1,
additionally comprising [0636] (c) a Fc region composed of a first
and a second subunit capable of stable association.
[0637] 3. The bispecific antigen binding molecule of para 1 or para
2, wherein the antigen binding domain capable of specific binding
to CD40 binds to a polypeptide comprising, or consisting of, the
amino acid sequence of SEQ ID NO:1.
[0638] 4. The bispecific antigen binding molecule of any one of
paras 1 to 3, wherein the antigen binding domain capable of
specific binding to a target cell antigen is an antigen binding
domain capable of specific binding to Fibroblast Activation Protein
(FAP).
[0639] 5. The bispecific antigen binding molecule of para 1 or para
2, wherein the antigen binding domain capable of specific binding
to FAP binds to a polypeptide comprising, or consisting of, the
amino acid sequence of SEQ ID NO:2.
[0640] 6. The bispecific antigen binding molecule of any one of
paras 1 to 5, wherein the antigen binding domain capable of
specific binding to FAP comprises
[0641] (a) a heavy chain variable region (V.sub.HFAP) comprising
(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (ii)
CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (iii)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:5, and a
light chain variable region (V.sub.LFAP) comprising (iv) CDR-L1
comprising the amino acid sequence of SEQ ID NO:6, (v) CDR-L2
comprising the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3
comprising the amino acid sequence of SEQ ID NO:8, or [0642] (b) a
heavy chain variable region (V.sub.HFAP) comprising (i) CDR-H1
comprising the amino acid sequence of SEQ ID NO:11, (ii) CDR-H2
comprising the amino acid sequence of SEQ ID NO:12, and (iii)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:13, and a a
light chain variable region (V.sub.LFAP) comprising (iv) CDR-L1
comprising the amino acid sequence of SEQ ID NO:14, (v) CDR-L2
comprising the amino acid sequence of SEQ ID NO:15, and (vi) CDR-L3
comprising the amino acid sequence of SEQ ID NO:16.
[0643] 7. The bispecific antigen binding molecule of any one of
paras 1 to 6, wherein the antigen binding domain capable of
specific binding to FAP comprises [0644] (a) a heavy chain variable
region (V.sub.HFAP) comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO:9, and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:10, or [0645] (b) a heavy chain variable
region (V.sub.HFAP) comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO:17, and a light chain variable region
(V.sub.LFAP) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO:18.
[0646] 8. The bispecific antigen binding molecule of any one of
paras 1 to 7, wherein the antigen binding domain capable of
specific binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising (i) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:19, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:20, and (iii) CDR-H3 comprising the amino
acid sequence of SEQ ID NO:21, and a light chain variable region
(V.sub.LCD40) comprising (iv) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:22, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:23, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:24.
[0647] 9. The bispecific antigen binding molecule of any one of
paras 1 to 7, wherein the antigen binding domain capable of
specific binding to CD40 comprises a heavy chain variable region
(V.sub.HCD40) comprising (i) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:27, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:28, and (iii) CDR-H3 comprising the amino
acid sequence of SEQ ID NO:29, and a light chain variable region
(V.sub.LCD40) comprising (iv) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:30, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:31, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:32.
[0648] 10. The bispecific antigen binding molecule of any one of
paras 1 to 9, wherein the antigen binding domain capable of
specific binding to CD40 comprises
[0649] (a) a VH comprising the amino acid sequence of SEQ ID NO:25
and a VL comprising the amino acid sequence of SEQ ID NO:26, or
[0650] (b) a VH comprising the amino acid sequence of SEQ ID NO:33
and a VL comprising the amino acid sequence of SEQ ID NO:34.
[0651] 11. The bispecific antigen binding molecule of any one of
paras 1 to 8, wherein the antigen binding domain capable of
specific binding to CD40 comprises
[0652] (i) a heavy chain variable region (V.sub.HCD40) comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ
ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54
and SEQ ID NO:55, and
[0653] (ii) a light chain variable region (V.sub.LCD40) comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,
SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64.
[0654] 12. The bispecific antigen binding molecule of any one of
paras 1 to 8 or 11, wherein the antigen binding domain capable of
specific binding to CD40 comprises a VH comprising the amino acid
sequence of SEQ ID NO:47 and a VL comprising the amino acid
sequence of SEQ ID NO:57.
[0655] 13. The bispecific antigen binding molecule of any one of
paras 1 to 8, comprising
[0656] (i) at least one antigen binding domain capable of specific
binding to CD40, comprising a heavy chain variable region
(V.sub.HCD40) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:25, SEQ ID NO:45, SEQ ID NO:46, SEQ
ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, and a
light chain variable region (V.sub.LCD40) comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:26, SEQ ID
NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ
ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, and
[0657] (ii) at least one antigen binding domain capable of specific
binding to FAP, comprising a heavy chain variable region
(V.sub.LFAP) comprising an amino acid sequence of SEQ ID NO:9 and a
light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:10, or a heavy chain variable region
(V.sub.LFAP) comprising an amino acid sequence of SEQ ID NO:17 and
a light chain variable region (V.sub.LFAP) comprising an amino acid
sequence of SEQ ID NO:18.
[0658] 14. The bispecific antigen binding molecule of any one of
paras 2 to 13, wherein the Fc region is an IgG, particularly an
IgG1 Fc region or an IgG4 Fc region.
[0659] 15. The bispecific antigen binding molecule of any one of
paras 2 to 14, wherein the Fc region comprises one or more amino
acid substitution that reduces the binding affinity of the antibody
to an Fc receptor and/or effector function.
[0660] 16. The bispecific antigen binding molecule of any one of
paras 2 to 15, wherein the Fc region is (i) of human IgG1 subclass
with the amino acid mutations L234A, L235A and P329G (numbering
according to Kabat EU index), or (ii) of mouse IgG1 subclass with
the amino acid mutations D265A and P329G (numbering according to
Kabat EU index).
[0661] 17. The bispecific antigen binding molecule of any one of
paras 2 to 16, wherein the Fc region comprises a modification
promoting the association of the first and second subunit of the Fc
region.
[0662] 18. The bispecific antigen binding molecule of any one of
paras 2 to 17, wherein the first subunit of the Fc region comprises
knobs and the second subunit of the Fc region comprises holes
according to the knobs into holes method.
[0663] 19. The bispecific antibody of any one of paras 2 to 18,
wherein
[0664] (i) the first subunit of the Fc region comprises the amino
acid substitutions S354C and T366W (numbering according to Kabat EU
index) and the second subunit of the Fc region comprises the amino
acid substitutions Y349C, T366S and Y407V (numbering according to
Kabat EU index), or
[0665] (ii) the first subunit of the Fc region comprises the amino
acid substitutions K392D and K409D (numbering according to Kabat EU
index) and the second subunit of the Fc region comprises the amino
acid substitutions E356K and:D.sup.-399K (numbering according to
Kabat EU index).
[0666] 20. The bispecific antigen binding molecule of any one of
paras 1 to 19, wherein the bispecific antigen binding molecule
comprises
[0667] (a) at least two Fab fragments capable of specific binding
to CD40 connected to a Fc region, and
[0668] (b) at least one antigen binding domain capable of specific
binding to FAP connected to the C-terminus of the Fc region.
[0669] 21. The bispecific antigen binding molecule of any one of
paras 1 to 19, wherein the bispecific antigen binding molecule
comprises
[0670] (a) two light chains and two heavy chains of an antibody
comprising two Fab fragments capable of specific binding to CD40,
and a Fc region, and
[0671] (b) a VH and a VL of an antigen binding domain capable
specific binding to FAP, wherein the VH is connected to the
C-terminus of one of the two heavy chains of (a), and wherein the
VL is connected to the C-terminus of the other of the two heavy
chains of (a).
[0672] 22. The bispecific antigen binding molecule of any one of
paras 1 to 19, wherein the bispecific antigen binding molecule
comprises
[0673] (a) two light chains and two heavy chains of an antibody
comprising two Fab fragments capable of specific binding to CD40,
and a Fc region, and
[0674] (b) two Fab fragments capable of specific binding to FAP,
wherein one of the Fab fragments is connected to the C-terminus of
one of the two heavy chains of (a), and the other of the Fab
fragments is connected to the C-terminus of the other of the two
heavy chains of (a).
[0675] 23. The bispecific antigen binding molecule of any one of
paras 1 to 19, wherein the bispecific antigen binding molecule
comprises
[0676] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40
and a Fc region subunit,
[0677] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0678] (c) a VH and a VL of an antigen binding domain capable of
specific binding to FAP, wherein the VH is connected to the
C-terminus of one of the two heavy chains of (a), and wherein the
VL is connected to the C-terminus of the other of the two heavy
chains of (a).
[0679] 24. The bispecific antigen binding molecule of any one of
paras 1 to 19, wherein the bispecific antigen binding molecule
comprises
[0680] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40,
and a Fc region subunit,
[0681] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0682] (c) two Fab fragments capable of specific binding to FAP,
wherein one of the Fab fragments is connected to the C-terminus of
one of the two heavy chains of (a), and the other of the Fab
fragments is connected to the C-terminus of the other of the two
heavy chains of (a).
[0683] 25. The bispecific antigen binding molecule of any one of
paras 1 to 19, wherein the bispecific antigen binding molecule
comprises
[0684] (a) two heavy chains, each heavy chain comprising a VH and
CH1 domain of a Fab fragment capable of specific binding to CD40,
and a Fc region subunit,
[0685] (b) two light chains, each light chain comprising a VL and
CL domain of a Fab fragment capable of specific binding to CD40,
and
[0686] (c) one Fab fragment capable of specific binding to FAP,
wherein the Fab fragments is connected to the C-terminus of one of
the two heavy chains of (a).
[0687] 26. The bispecific antigen binding molecule of any one of
paras 22 to 25, wherein the Fab fragment or the two Fab fragments
capable of specific binding to FAP are crossover Fab fragments each
comprising a VL-CH1 chain and a VH-CL chain, and wherein the VL-CH1
chain is connected to the C-terminus of one of the two heavy chains
of (a).
[0688] 27. The bispecific antigen binding molecule of any one of
paras 1 to 26, wherein the bispecific antigen binding molecule
comprises four Fab fragments capable of specific binding to
CD40.
[0689] 28. The bispecific antigen binding molecule of any one of
paras 23 to 27, wherein each of the two heavy chains of (a)
comprises two VHs and two CH1 domains of a Fab fragment capable of
specific binding to CD40.
[0690] 29. The bispecific antigen binding molecule of any one of
paras 23 to 28, wherein one or more of the Fab fragments capable of
specific binding to CD40 comprises
[0691] a CL domain comprising an arginine (R) at amino acid at
position 123 (numbering according to Kabat EU index) and a lysine
(K) at amino acid at position 124 (numbering according to Kabat EU
index), and
[0692] a CH1 domain comprising a glutamic acid (E) at amino acid at
position 147 (numbering according to Kabat EU index) and a glutamic
acid (E) at amino acid at position 213 (numbering according to
Kabat EU index).
[0693] 30. A polynucleotide encoding the bispecific antigen binding
molecule of any one of paras 1 to 29.
[0694] 31. An expression vector comprising the polynucleotide of
claim 30.
[0695] 32. A host cell comprising the polynucleotide of para 30 or
the expression vector of para 31.
[0696] 33. A method of producing a bispecific antigen binding
molecule, comprising culturing the host cell of para 32 under
conditions suitable for the expression of the bispecific antigen
binding molecule, and isolating the bispecific antigen binding
molecule.
[0697] 34. A pharmaceutical composition comprising the bispecific
antigen binding molecule of any one of paras 1 to 29 and at least
one pharmaceutically acceptable excipient.
[0698] 35. The bispecific antigen binding molecule of any one of
paras 1 to 29, or the pharmaceutical composition of para 34, for
use as a medicament.
[0699] 36. The bispecific antigen binding molecule of any one of
paras 1 to 29, or the pharmaceutical composition of para 34, for
use [0700] (i) in inducing immune stimulation by CD40.sup.+
antigen-presenting cells (APCs), [0701] (ii) in stimulating
tumor-specific T cell response, [0702] (iii) in causing apoptosis
of tumor cells, [0703] (iv) in the treatment of cancer, [0704] (v)
in delaying progression of cancer, [0705] (vi) in prolonging the
survival of a patient suffering from cancer, [0706] (vii) in the
treatment of infections.
[0707] 37. The bispecific antigen binding molecule of any one of
paras 1 to 29, or the pharmaceutical composition of para 34, for
use in the treatment of cancer.
[0708] 38. Use of the bispecific antigen binding molecule of any
one of paras 1 to 29, or the pharmaceutical composition of para 34,
in the manufacture of a medicament for the treatment of cancer.
[0709] 39. A method of treating an individual having cancer
comprising administering to the individual an effective amount of
the bispecific antigen binding molecule of any one of paras 1 to
29, or the pharmaceutical composition of para 34.
[0710] 40. The bispecific antigen binding molecule of any one of
paras 1 to 29, or the pharmaceutical composition of para 34, for
use in up-regulating or prolonging cytotoxic T cell activity.
[0711] 41. The bispecific antigen binding molecule according to any
one of paras 1 to 29 or the pharmaceutical composition according to
para 34 for use in the treatment of cancer, wherein the bispecific
antigen binding molecule is administered in combination with a
chemotherapeutic agent, radiation and/or other agents for use in
cancer immunotherapy.
EXAMPLES
[0712] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
Recombinant DNA Techniques
[0713] Standard methods were used to manipulate DNA as described in
Sambrook et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions. General information regarding the
nucleotide sequences of human immunoglobulin light and heavy chains
is given in: Kabat, E. A. et al., (1991) Sequences of Proteins of
Immunological Interest, Fifth Ed., NIH Publication No 91-3242.
DNA Sequencing
[0714] DNA sequences were determined by double strand
sequencing.
Gene Synthesis
[0715] Desired gene segments were either generated by PCR using
appropriate templates or were synthesized by Geneart AG
(Regensburg, Germany) from synthetic oligonucleotides and PCR
products by automated gene synthesis. In cases where no exact gene
sequence was available, oligonucleotide primers were designed based
on sequences from closest homologues and the genes were isolated by
RT-PCR from RNA originating from the appropriate tissue. The gene
segments flanked by singular restriction endonuclease cleavage
sites were cloned into standard cloning/sequencing vectors. The
plasmid DNA was purified from transformed bacteria and
concentration determined by UV spectroscopy. The DNA sequence of
the subcloned gene fragments was confirmed by DNA sequencing. Gene
segments were designed with suitable restriction sites to allow
sub-cloning into the respective expression vectors. All constructs
were designed with a 5'-end DNA sequence coding for a leader
peptide which targets proteins for secretion in eukaryotic
cells.
Protein Purification
[0716] Proteins were purified from filtered cell culture
supernatants referring to standard protocols. In brief, antibodies
were applied to a Protein A Sepharose column (GE healthcare) and
washed with PBS. Elution of antibodies was achieved at pH 2.8
followed by immediate neutralization of the sample. Aggregated
protein was separated from monomeric antibodies by size exclusion
chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM
Histidine, 150 mM NaCl pH 6.0. Monomeric antibody fractions were
pooled, concentrated (if required) using e.g., a MILLIPORE Amicon
Ultra (30 MWCO) centrifugal concentrator, frozen and stored at
-20.degree. C. or -80.degree. C. Part of the samples were provided
for subsequent protein analytics and analytical characterization
e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass
spectrometry.
SDS-PAGE
[0717] The NuPAGE.RTM. Pre-Cast gel system (Invitrogen) was used
according to the manufacturer's instruction. In particular, 10% or
4-12% NuPAGE.RTM. Novex.RTM. Bis-TRIS Pre-Cast gels (pH 6.4) and a
NuPAGE.RTM. MES (reduced gels, with NuPAGE.RTM. Antioxidant running
buffer additive) or MOPS (non-reduced gels) running buffer was
used.
CE-SDS
[0718] Purity, antibody integrity and molecular weight of
bispecific and control antibodies were analyzed by CE-SDS using
microfluidic Labchip technology (Caliper Life Science, USA). 5
.mu.l of protein solution was prepared for CE-SDS analysis using
the HT Protein Express Reagent Kit according manufacturer's
instructions and analysed on LabChip GXII system using a HT Protein
Express Chip. Data were analyzed using LabChip GX Software version
3.0.618.0.
Analytical Size Exclusion Chromatography
[0719] Size exclusion chromatography (SEC) for the determination of
the aggregation and oligomeric state of antibodies was performed by
HPLC chromatography. Briefly, Protein A purified antibodies were
applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM
KH.sub.2PO.sub.4/K.sub.2HPO.sub.4, pH 7.5 on an Agilent HPLC 1100
system or to a Superdex 200 column (GE Healthcare) in 2.times. PBS
on a Dionex HPLC-System. The eluted protein was quantified by UV
absorbance and integration of peak areas. BioRad Gel Filtration
Standard 151-1901 served as a standard.
Mass Spectrometry
[0720] This section describes the characterization of the
multispecific antibodies with VH/VL or CH/CL exchange (CrossMabs)
with emphasis on their correct assembly. The expected primary
structures were analyzed by electrospray ionization mass
spectrometry (ESI-MS) of the deglycosylated intact CrossMabs and
deglycosylated/FabALACTICA or alternatively
deglycosylated/GingisKHAN digested CrossMabs.
[0721] The CrossMabs were deglycosylated with N-Glycosidase F in a
phosphate or Tris buffer at 37.degree. C. for up to 17 h at a
protein concentration of 1 mg/ml. The FabALACTICA or GingisKHAN
(Genovis AB; Sweden) digestions were performed in the buffers
supplied by the vendor with 100 .mu.g deglycosylated CrossMabs.
Prior to mass spectrometry the samples were desalted via HPLC on a
Sephadex G25 column (GE Healthcare). The total mass was determined
via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik)
equipped with a TriVersa NanoMate source (Advion).
Example 1
Generation and Production of Bispecific Constructs Targeting CD40
and Fibroblast Activation Protein (FAP)
1.1 Generation of Bispecific Antigen Binding Molecules Targeting
CD40 and Fibroblast Activation Protein (FAP)
[0722] The cDNAs encoding variable heavy and light chain domains of
the anti CD40 binder (SEQ ID NO:10 and SEQ ID NO:16 of WO
2006/128103) were cloned in frame with the corresponding constant
heavy or light chains of human IgG1 in suitable expression
plasmids. Expression of heavy and light chain is driven by a
chimeric MPSV promoter consisting of the MPSV core promoter and a
CMV enhancer element. The expression cassette also contains a
synthetic polyA signal at the 3' end of the cDNAs. In addition the
plasmid vectors harbor an origin of replication (EBV OriP) for
episomal maintenance of the plasmids. Amino acid and nucleotide
sequences of the variable domains of the CD40 mAb and the FAP mAb
are shown in Table 1 and 2, respectively.
[0723] Different bispecific CD40-FAP antibodies have been prepared
in 4+1 and 4+2 formats consisting of four CD40 binding moieties
combined with either one or two FAP binding arms at the C-terminus
of an Fc or in 2+1 and 2+2 formats consisting of two CD40 binding
moieties combined with either one or two FAP binding arms at the
C-terminus of an Fc (FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D). The
generation and preparation of FAP binder 28H1 is described in WO
2012/020006 A2, which is incorporated herein by reference. To
generate the 4+1 and the 2+1 molecules the knob-into-hole
technology was used to achieve heterodimerization. The S354C/T366W
mutations were introduced in the first heavy chain HC1 (Fc knob
heavy chain) and the Y349C/T366S/L368A/Y407V mutations were
introduced in the second heavy chain HC2 (Fc hole heavy chain). In
the 4+2 and 2+2 molecules the CrossMab technology as described in
WO 2010/145792 Al ensured correct light chain pairing. Independent
of the bispecific format, in all cases an effector silent Fc
(P329G; L234, L235A) was used to abrogate binding to Fc.gamma.
receptors according to the method described in WO 2012/130831 A1.
Sequences of the bispecific molecules are shown in Table 3 and
4.
[0724] Besides molecules targeting the human receptors also
surrogate molecules were generated in the same formats that
recognize the murine antigens. In these cases the
heterodimerization of 4+1 molecules was achieved by DD/KK mutations
(introduction of the Lys392Asp and Lys409Asp in the first heavy
chain and introduction of the Glu356Lys and Asp399Lys mutation in
the second heavy chain) in the Fc according to the method described
in by Gunasekaran et al., J. Biol. Chem. 2010,19637-19646, while
binding to Fc receptors was inhibited by D270A/P329G mutations in
accordance with the method described in Baudino et al., J. Immunol.
(2008), 181, 6664-9. or in WO 2016/030350 A1.
[0725] All genes were transiently expressed under control of a
chimeric MPSV promoter consisting of the MPSV core promoter
combined with the CMV promoter enhancer fragment. The expression
vector also contains the oriP region for episomal replication in
EBNA (Epstein Barr Virus Nuclear Antigen) containing host
cells.
TABLE-US-00004 TABLE 1 Amino acid sequences of the variable domains
of the CD40 antibodies, the FAP antibody and DP47 antibody Seq ID
Description Sequence No hu CD40 VH
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 25
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTLVTVSS hu CD40 VL
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 26
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIK FAP 28H1 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 9
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSS FAP 28H1 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQ 10
APRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQGQVIPPTFGQGTKVEIK
DP47 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 71
GLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCAKGSGFDYWGQGTLVTVSS DP47 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQ 72
APRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQYGSSPLTFGQGTKVEIK
mu CD40 VH EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 33
GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL
RSEDTATYYCGRHSSYFDYWGQGVMVTVSS mu CD40 VL
DTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 34
KLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYY
CQQSWNDPWTFGGGTKLELK
TABLE-US-00005 TABLE 2 Nucleotide sequences of the variable domains
of the CD40 antibodies, the FAP antibody and DP47 antibody Seq ID
Description Sequence No hu CD40 VH
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 73
GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGC hu CD40 VL
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCG 74
TGGGCGACAGAGTGACCATCACCTGTCGGAGCAGCCAGAGCCT
GGTGCACAGCAACGGCAACACCTTCCTGCACTGGTATCAGCAG
AAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACACCGTGTCCA
ACCGGTTCAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTC
CGGCACCGACTTCACCCTGACAATCAGCTCCCTGCAGCCCGAG
GACTTCGCCACCTATTTCTGCAGCCAGACCACCCACGTGCCCT
GGACATTTGGACAGGGCACCAAGGTGGAAATCAAG FAP 28H1 VH
GAGGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTG 75
GCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTT
CTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAA
GGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGT
ACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGA
CAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGG
GCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGG
GCAACTTCGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTC CAGC FAP 28H1 VL
GAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCTGAGCC 76
CTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGT
GAGCCGGAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAG
GCCCCCAGACTGCTGATCATCGGCGCCAGCACCCGGGCCACCG
GCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTT
CACCCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTG
TACTACTGCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCC AGGGCACCAAGGTGGAAATCAAG
DP47 VH GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG 77
GGGGGTCCCTGAGACTCTCCTGTGCAGCCAGCGGATTCACCTT
TAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAG
AGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTG
AGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCG
GATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAG C DP47 VL
GAAATTGTGCTGACCCAGAGCCCCGGCACCCTGTCACTGTCTC 78
CAGGCGAAAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGT
GTCCAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCCGGACAG
GCCCCCAGACTGCTGATCTACGGCGCCTCTTCTAGAGCCACCG
GCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTT
CACCCTGACAATCAGCAGACTGGAACCCGAGGACTTTGCCGTG
TATTACTGCCAGCAGTACGGCAGCAGCCCCCTGACCTTTGGCC AGGGCACCAAGGTGGAAATCAAA
mu CD40 VH GAAGTGCAGCTGGTGGAATCCGACGGCGGACTGGTGCAGCCTG 79
GCAGATCTCTGAAGCTGCCTTGTGCCGCCTCCGGCTTCACCTT
CTCCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG
GGACTGGAATGGGTGGCCTCCATCTCCTACGACGGCTCCTCCA
CCTACTACCGGGACTCTGTGAAGGGCCGGTTCACCATCTCTCG
GGACAACGCCAAGTCCACCCTGTACCTGCAGATGGACTCCCTG
CGGAGCGAGGACACCGCTACCTACTACTGCGGCAGACACTCCT
CCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTC CTCT mu CD40 VL
GACACTGTACTGACCCAGTCTCCTGCTTTGGCTGTGTCTCCAG 80
GAGAGAGGGTTACCATCTCCTGTAGGGCCAGTGACAGTGTCAG
TACACTTATGCACTGGTACCAACAGAAACCAGGACAGCAACCC
AAACTCCTCATCTATCTAGCATCACACCTAGAATCTGGGGTCC
CTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT
CACCATTGATCCTGTGGAGGCTGATGACACTGCAACCTATTAC
TGTCAGCAGAGTTGGAATGATCCGTGGACGTTCGGTGGAGGCA CCAAGCTGGAATTGAAA
TABLE-US-00006 TABLE 3 Amino acid sequences of the CD40 IgG and the
bispecific antigen binding molecules Seq ID Construct Sequence No
P1AD4470 CD40 IgG CD40 IgG
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 81 Heavy chain
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG CD40
light DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 82 chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AD4453 CD40 x
FAP (28H1) (4 + 1) CD40 VHCH1-
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 83 CD40 VHCH1-
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fcknob_PGLALA-
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS 28H1 VH
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQA
PGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMN
SLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS CD40 VHCH1-
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 84 CD40 VHCH1-
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fchole_PGLALA-
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS 28H1 VL
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQK
PGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPED
FAVYYCQQGQVIPPTFGQGTKVEIK CD40 light
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 82 chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AD4455 CD40 x
FAP (28H1) (4 + 2) CD40 VHCH1-
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 85 CD40 VHCH1-
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fc_PGLALA-
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS 28H1 VLCH1
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ `EE`
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQK
PGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPED
FAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C CD40-light
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 86 chain;
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE ,RK`
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 28H1 VHCL
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 87
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC P1AA9641 CD40
x FAP (28H1) (2 + 1) CD40_Fc
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 88 hole_PGLALA_28H1
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL VL
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGT
LSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGAS
TRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIP PTFGQGTKVEIK
huCD40_Fcknob_PGLALA_28H1
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 89 VH
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGG
LVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWA
SGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KGWLGNFDYWGQGTLVTVSS +
CD40 LC DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 82
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AA9663 CD40 x
FAP (28H1) (2 + 2) CD40-28H1
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 90 2 + 2; ,EE`
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL huCD40-
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS Fc_PGLALA_28H1_VLCH1
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ `EE`
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGT
LSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGAS
TRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIP
PTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSC + CD40
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 86 LC; ,RK`
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC + 28H1 VHCL
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 87
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC P1AD4574 CD40
x DP47 (4 + 1) CD40 VHCH1-
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 91 CD40 VHCH1-
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fcknob_PGLALA-
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS DP47 VH
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQM
NSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS CD40 VHCH1-
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 92 CD40 VHCH1-
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fchole_PGLALA-
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS DP47 VL
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK
PGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPED
FAVYYCQQYGSSPLTFGQGTKVEIK + CD40 light
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 82 chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AD4465 CD40 x
DP47 (4 + 2) CD40 VHCH1-
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 93 CD40 VHCH1-
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL Fc_PGLALA-
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS DP47 VLCH1
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ `EE`
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK
PGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPED
FAVYYCQQYGSSPLTFGQGTKVEIKSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C + CD40 light
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 86 chain; `RK`
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC + DP47VHCL
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 94
GLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCAKGSGFDYWGQGTLVTVSSASVAAPSVFIFPPS
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC P1AD4520 or
P1AD9139 mu CD40 (FGK4.5) x FAP (28H1) (4 + 1) muCD40
EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 95 VHCH1-
GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40
RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-
GSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV FcKK_DAPG-
LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP 28H1 VH
RDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFS
DYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRD
NAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSS
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNS
GSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAH
PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT
ITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQ
INSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISK
TKGRPKAPQVYTIPPPKKQMAKDKVSLTCMITNFFPEDITVEW
QWNGQPAENYKNTQPIMKTDGSYFVYSKLNVQKSNWEAGNTFT
CSVLHEGLHNHHTEKSLSHSPGGGGGSGGGGSGGGGSGGGGSE
VQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKG
LEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRA
EDTAVYYCAKGWLGNFDYWGQGTLVTVSS muCD40
EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 96 VHCH1-
GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40
RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-
GSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV FcDD_DAPG-
LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP 28H1 VL
RDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFS
DYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRD
NAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSS
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNS
GSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAH
PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT
ITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQ
INSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISK
TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEW
QWNGQPAENYDNTQPIMDTDGSYFVYSDLNVQKSNWEAGNTFT
CSVLHEGLHNHHTEKSLSHSPGGGGGSGGGGSGGGGSGGGGSE
IVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQA
PRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQGQVIPPTFGQGTKVEIK
mu CD40 light DTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 97 chain
KLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYY
CQQSWNDPWTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGA
SVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY
SMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC P1AD4558 mu CD40 (FGK5.4)
x FAP (28H1) (4 + 2) muCD40
EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 98 VHCH1-
GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40
RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-
GSAAQTNSMVTLGCLVEGYFPEPVTVTWNSGSLSSGVHTFPAV Fc_DAPG-28H1
LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDEKIVP VLCH1 `EE`
RDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFS
DYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRD
NAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSS
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVEGYFPEPVTVTWNS
GSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAH
PASSTKVDEKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT
ITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQ
INSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISK
TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEW
QWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFT
CSVLHEGLHNHHTEKSLSHSPGKGGGGSGGGGSGGGGSGGGGS
EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQ
APRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV
YYCQQGQVIPPTFGQGTKVEIKSSAKTTPPSVYPLAPGSAAQT
NSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY
TLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDC 28H1 VHCL
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 99 (mu)
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASDAAPTVSIFPP
SSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSW
TDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNEC mu CD40 light
DTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 100 chain; `RK`
KLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYY
CQQSWNDPWTFGGGTKLELKRADAAPTVSIFPPSSRKLTSGGA
SVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY
SMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC P1AD4521 mu CD40-DP47 (4
+ 1) muCD40 EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 101 VHCH1-
GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40
RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-
GSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV FcKK_DAPG-
LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP DP47 VH
RDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFS
DYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRD
NAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSS
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNS
GSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAH
PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT
ITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQ
INSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISK
TKGRPKAPQVYTIPPPKKQMAKDKVSLTCMITNFFPEDITVEW
QWNGQPAENYKNTQPIMKTDGSYFVYSKLNVQKSNWEAGNTFT
CSVLHEGLHNHHTEKSLSHSPGGGGGSGGGGSGGGGSGGGGSE
VQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKG
LEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGSGFDYWGQGTLVTVSS muCD40
EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 102 VHCH1-mu
GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL CD40 VHCH1-
RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP FcDD_DAPG-
GSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV DP47 VL
LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP
RDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFS
DYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRD
NAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSS
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNS
GSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAH
PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT
ITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQ
INSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISK
TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEW
QWNGQPAENYDNTQPIMDTDGSYFVYSDLNVQKSNWEAGNTFT
CSVLHEGLHNHHTEKSLSHSPGGGGGSGGGGSGGGGSGGGGSE
IVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQA
PRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQYGSSPLTFGQGTKVEIK +
mu CD40 DTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 97 light chain
KLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYY
CQQSWNDPWTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGA
SVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY
SMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC P1AD4555 mu CD40-DP47 (4
+ 2) muCD40 EVQLVESDGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTK 103 VHCH1-
GLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSL muCD40
RSEDTATYYCGRHSSYFDYWGQGVMVTVSSAKTTPPSVYPLAP VHCH1-
GSAAQTNSMVTLGCLVEGYFPEPVTVTWNSGSLSSGVHTFPAV Fc_DAPG-
LQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDEKIVP DP47 VLCH1
RDCGGGGSGGGGSEVQLVESDGGLVQPGRSLKLPCAASGFTFS `EE`
DYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRD
NAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGVMVTVSS
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVEGYFPEPVTVTWNS
GSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAH
PASSTKVDEKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT
ITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQ
INSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISK
TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEW
QWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFT
CSVLHEGLHNHHTEKSLSHSPGKGGGGSGGGGSGGGGSGGGGS
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQ
APRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV
YYCQQYGSSPLTFGQGTKVEIKSSAKTTPPSVYPLAPGSAAQT
NSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY
TLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDC + mu CD40
DTVLTQSPALAVSPGERVTISCRASDSVSTLMHWYQQKPGQQP 100 light chain;
KLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYY ,RK`
CQQSWNDPWTFGGGTKLELKRADAAPTVSIFPPSSRKLTSGGA
SVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY
SMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC DP47 VHCL
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 104 (mu)
GLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCAKGSGFDYWGQGTLVTVSSASDAAPTVSIFPPS
SEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWT
DQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVK SFNRNEC
TABLE-US-00007 TABLE 4 Nucleotide sequences of the bispecific
antigen binding molecules Seq ID Construct Sequence No CD40 IgG
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 105 Heavy chain
GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGC
TAGCACCAAGGGCCCAAGCGTGTTCCCACTGGCCCCAAGCAGC
AAGTCTACCAGCGGAGGAACAGCCGCCCTGGGATGTCTGGTGA
AGGACTACTTCCCCGAGCCAGTGACAGTGAGCTGGAACTCTGG
CGCCCTGACATCTGGCGTGCACACATTCCCAGCCGTGCTGCAG
TCTAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCAA
GCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCA
CAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAG
AGCTGCGACAAGACCCACACCTGCCCACCATGTCCAGCCCCAG
AGCTGCTGGGAGGACCTAGCGTGTTCCTGTTCCCCCCCAAGCC
AAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACATGT
GTGGTGGTGGACGTGTCTCACGAGGACCCAGAGGTGAAGTTCA
ACTGGTACGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAA
GCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCT
GTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAAT
ACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCAATCGA
AAAGACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAGCCACAG
GTGTACACCCTGCCCCCATCTAGGGAGGAGATGACCAAGAACC
AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA
CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC
TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD40 light
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCG 106 chain
TGGGCGACAGAGTGACCATCACCTGTCGGAGCAGCCAGAGCCT
GGTGCACAGCAACGGCAACACCTTCCTGCACTGGTATCAGCAG
AAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACACCGTGTCCA
ACCGGTTCAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTC
CGGCACCGACTTCACCCTGACAATCAGCTCCCTGCAGCCCGAG
GACTTCGCCACCTATTTCTGCAGCCAGACCACCCACGTGCCCT
GGACATTTGGACAGGGCACCAAGGTGGAAATCAAGCGTACGGT
GGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAG
TTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT
TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGC
CCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA
GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT
CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC AGGGGAGAGTGT CD40 x FAP
(28H1) (4 + 1) CD40 GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 107
VHCH1-CD40 GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fchole_PGLALA-
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA 28H1 VH
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGC
TTCCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGC
AAGTCTACCAGCGGAGGAACAGCCGCCCTGGGCTGCCTCGTGA
AGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG
CGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAG
AGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCA
GCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCA
CAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAG
AGCTGCGACGGCGGAGGCGGATCAGGCGGCGGAGGATCCGAAG
TGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGG
AAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACC
GGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCC
TGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTC
TTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGAC
AATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCG
CCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTA
TTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGC
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGA
GCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC
CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG
CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG
CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGC
TGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG
CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC
TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGT
GTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATC
GCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACA
AGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCT
GTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGC
AACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACC
ACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGG
CGGAAGCGGAGGAGGAGGATCCGGAGGAGGGGGAAGTGGCGGC
GGAGGATCTGAGGTGCAGCTGCTGGAATCCGGCGGAGGCCTGG
TGCAGCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGG
CTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGCT
CCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCG
GCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCAT
CTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAAC
TCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGG
GCTGGCTGGGCAACTTCGACTACTGGGGCCAGGGCACCCTGGT CACCGTGTCCAGC CD40
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 108 VHCH1-CD40
GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fchole_PGLALA-
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA 28H1 VL
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGC
TTCCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGC
AAGTCTACCAGCGGAGGAACAGCCGCCCTGGGCTGCCTCGTGA
AGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG
CGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAG
AGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCA
GCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCA
CAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAG
AGCTGCGACGGCGGAGGCGGATCAGGCGGCGGAGGATCCGAAG
TGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGG
AAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACC
GGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCC
TGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTC
TTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGAC
AATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCG
CCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTA
TTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGC
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGA
GCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC
CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG
CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG
CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGC
TGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG
CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC
TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
GCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT
CAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT
CGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG
AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGG
CGGAAGCGGAGGAGGAGGATCCGGTGGTGGCGGATCTGGGGGC
GGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGA
GCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAG
CCAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCAGAAG
CCCGGCCAGGCCCCCAGACTGCTGATCATCGGCGCCAGCACCC
GGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGG
CACCGACTTCACCCTGACCATCAGCCGGCTGGAACCCGAGGAC
TTCGCCGTGTACTACTGCCAGCAGGGCCAGGTGATCCCCCCCA
CCTTCGGCCAGGGCACCAAGGTGGAAATCAAG CD40 light see above 106 chain
CD40 x FAP (28H1) (4 + 2) CD40
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 109 VHCH1-CD40
GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fc_PGLALA-
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA 28H1 VLCH1
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT `EE`
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGC
TTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGC
AAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGG
AGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG
CGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTCCAG
AGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCA
GCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCA
CAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAACCCAAG
AGCTGCGACGGCGGAGGCGGATCTGGCGGCGGAGGATCCGAAG
TGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGG
AAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACC
GGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCC
TGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTC
TTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGAC
AATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCG
CCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTA
TTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGC
ACCAAGGGACCTTCCGTGTTTCCCCTGGCTCCCAGCTCCAAGT
CTACCTCTGGGGGCACAGCTGCTCTGGGATGTCTGGTGGAAGA
TTATTTTCCTGAACCTGTGACCGTGTCATGGAACAGCGGAGCC
CTGACCTCCGGGGTGCACACATTCCCTGCTGTGCTGCAGTCCT
CCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCAG
CTCTCTGGGCACACAGACATATATCTGTAATGTGAATCACAAA
CCCTCTAATACCAAAGTGGATGAGAAAGTGGAACCTAAGTCCT
GCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGC
TGCTGGCGGCCCATCTGTGTTTCTGTTCCCCCCAAAGCCCAAG
GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGG
TGGTGGATGTGTCCCACGAGGACCCAGAAGTGAAGTTCAATTG
GTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCG
CGGGAAGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGC
TGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAA
GTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAGAAA
ACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCTCAGGTGT
ACACCCTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGT
GTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATC
GCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACA
AGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCT
GTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGC
AACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACC
ACTACACACAGAAGTCTCTGAGCCTGAGCCCTGGCGGAGGGGG
AGGATCTGGGGGAGGCGGAAGTGGGGGAGGGGGTTCCGGAGGC
GGCGGATCAGAAATTGTGCTGACCCAGTCCCCCGGCACCCTGT
CACTGTCTCCAGGCGAAAGAGCCACCCTGAGCTGTAGGGCCTC
CCAGAGCGTGTCCAGAAGCTATCTGGCCTGGTATCAGCAGAAG
CCCGGACAGGCCCCCAGACTGCTGATCATTGGCGCCTCTACCA
GAGCCACCGGCATCCCCGATAGATTCAGCGGCTCTGGCAGCGG
CACCGACTTCACCCTGACCATCTCCAGACTGGAACCCGAGGAC
TTTGCCGTGTACTATTGCCAGCAGGGCCAAGTGATCCCCCCCA
CCTTTGGCCAGGGAACAAAGGTGGAAATCAAGTCCAGCGCTTC
CACCAAGGGCCCCTCAGTGTTCCCACTGGCACCATCCAGCAAG
TCCACAAGCGGAGGAACCGCCGCTCTGGGCTGTCTCGTGAAAG
ACTACTTTCCAGAGCCAGTGACCGTGTCCTGGAATAGTGGCGC
TCTGACTTCTGGCGTGCACACTTTCCCCGCAGTGCTGCAGAGT
TCTGGCCTGTACTCCCTGAGTAGCGTCGTGACAGTGCCCTCCT
CTAGCCTGGGCACTCAGACTTACATCTGCAATGTGAATCATAA
GCCTTCCAACACAAAAGTGGACAAAAAAGTGGAACCCAAATCT TGC CD40-light
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCG 110 chain;
TGGGCGACAGAGTGACCATCACCTGTCGGAGCAGCCAGAGCCT ,RK`
GGTGCACAGCAACGGCAACACCTTCCTGCACTGGTATCAGCAG
AAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACACCGTGTCCA
ACCGGTTCAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTC
CGGCACCGACTTCACCCTGACAATCAGCTCCCTGCAGCCCGAG
GACTTCGCCACCTATTTCTGCAGCCAGACCACCCACGTGCCCT
GGACATTTGGACAGGGCACCAAGGTGGAAATCAAGCGTACGGT
GGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATCGGAAG
TTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT
TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGC
CCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA
GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT
CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC AGGGGAGAGTGT 28H1 VHCL
GAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTG 111
GCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTT
CTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAA
GGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGT
ACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGA
CAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGG
GCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGG
GCAACTTCGACTACTGGGGACAGGGCACCCTGGTCACCGTGTC
CAGCGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCT
TCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCC
TGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAA
GGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTG
ACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCA
CCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTA
CGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACC
AAGTCTTTCAACCGGGGCGAGTGC CD40 x FAP (28H1) (2 + 1) pETR17111
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 112
huCD40_Fchole_PGLALA_28H1
GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VL
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGC
TAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC
AAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG
CGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG
TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT
CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAA
TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT
ACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACC
AGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGA
CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC
TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAG
GCGGCGGAAGCGGAGGAGGAGGATCCGGTGGTGGCGGATCTGG
GGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACC
CTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAG
CCAGCCAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCA
GAAGCCCGGCCAGGCCCCCAGACTGCTGATCATCGGCGCCAGC
ACCCGGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCT
CCGGCACCGACTTCACCCTGACCATCAGCCGGCTGGAACCCGA
GGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTGATCCCC
CCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAGTGA pETR17112
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 113
huCD40_Fcknob_PGLALA_28H1
GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VH
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGC
TAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC
AAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG
CGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG
TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT
CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAA
TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT
ACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACC
AGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGA
TATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAAC
TACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCT
TCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCA
GGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC
AACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAG
GCGGCGGAAGCGGAGGAGGAGGATCCGGAGGAGGGGGAAGTGG
CGGCGGAGGATCTGAGGTGCAGCTGCTGGAATCCGGCGGAGGC
CTGGTGCAGCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCT
CCGGCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACA
GGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCC
TCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCA
CCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGAT
GAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCC
AAGGGCTGGCTGGGCAACTTCGACTACTGGGGCCAGGGCACCC TGGTCACCGTGTCCAGCTGA
+CD40 LC see above 106 pETR15390 CD40 x FAP (28H1) (2 + 2)
pETR17113 GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 114 huCD40-
GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT Fc_PGLALA_28H1_VLCH1
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG `EE`
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGC
TAGCACCAAGGGACCTTCCGTGTTTCCCCTGGCTCCCAGCTCC
AAGTCTACCTCTGGGGGCACAGCTGCTCTGGGATGTCTGGTGG
AAGATTATTTTCCTGAACCTGTGACCGTGTCATGGAACAGCGG
AGCCCTGACCTCCGGGGTGCACACATTCCCTGCTGTGCTGCAG
TCCTCCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTT
CCAGCTCTCTGGGCACACAGACATATATCTGTAATGTGAATCA
CAAACCCTCTAATACCAAAGTGGATGAGAAAGTGGAACCTAAG
TCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTG
AAGCTGCTGGCGGCCCATCTGTGTTTCTGTTCCCCCCAAAGCC
CAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGC
GTGGTGGTGGATGTGTCCCACGAGGACCCAGAAGTGAAGTTCA
ATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAA
GCCGCGGGAAGAACAGTACAACAGCACCTACCGGGTGGTGTCC
GTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGT
ACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGA
GAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCTCAG
GTGTACACCCTGCCCCCAAGCAGGGACGAGCTGACCAAGAACC
AGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGA
TATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAAC
TACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCT
TCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCA
GGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC
AACCACTACACACAGAAGTCTCTGAGCCTGAGCCCTGGCGGAG
GGGGAGGATCTGGGGGAGGCGGAAGTGGGGGAGGGGGTTCCGG
AGGCGGCGGATCAGAAATTGTGCTGACCCAGTCCCCCGGCACC
CTGTCACTGTCTCCAGGCGAAAGAGCCACCCTGAGCTGTAGGG
CCTCCCAGAGCGTGTCCAGAAGCTATCTGGCCTGGTATCAGCA
GAAGCCCGGACAGGCCCCCAGACTGCTGATCATTGGCGCCTCT
ACCAGAGCCACCGGCATCCCCGATAGATTCAGCGGCTCTGGCA
GCGGCACCGACTTCACCCTGACCATCTCCAGACTGGAACCCGA
GGACTTTGCCGTGTACTATTGCCAGCAGGGCCAAGTGATCCCC
CCCACCTTTGGCCAGGGAACAAAGGTGGAAATCAAGTCCAGCG
CTTCCACCAAGGGCCCCTCAGTGTTCCCACTGGCACCATCCAG
CAAGTCCACAAGCGGAGGAACCGCCGCTCTGGGCTGTCTCGTG
AAAGACTACTTTCCAGAGCCAGTGACCGTGTCCTGGAATAGTG
GCGCTCTGACTTCTGGCGTGCACACTTTCCCCGCAGTGCTGCA
GAGTTCTGGCCTGTACTCCCTGAGTAGCGTCGTGACAGTGCCC
TCCTCTAGCCTGGGCACTCAGACTTACATCTGCAATGTGAATC
ATAAGCCTTCCAACACAAAAGTGGACAAAAAAGTGGAACCCAA ATCTTGCTGA +CD40 see
above 110 LC; ,RK` pETR15391 +28H1 VHCL see above 111 pETR15114
CD40 x DP47 (4 + 1) CD40
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 115 VHCH1-CD40
GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fcknob_PGLALA-
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA DP47 VH
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGC
TTCCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGC
AAGTCTACCAGCGGAGGAACAGCCGCCCTGGGCTGCCTCGTGA
AGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG
CGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAG
AGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCA
GCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCA
CAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAG
AGCTGCGACGGCGGAGGCGGATCAGGCGGCGGAGGATCCGAAG
TGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGG
AAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACC
GGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCC
TGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTC
TTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGAC
AATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCG
CCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTA
TTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGC
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGA
GCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC
CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG
CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG
CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGC
TGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG
CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC
TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGT
GTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATC
GCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACA
AGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCT
GTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGC
AACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACC
ACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGG
CGGAAGCGGAGGAGGAGGATCCGGAGGAGGGGGAAGTGGCGGC
GGAGGATCTGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGG
TACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCAGCGG
ATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCT
CCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTG
GTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATG
AACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGA
AAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCAC CGTCTCGAGC CD40
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCG 116 VHCH1-CD40
GCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTACAGCTT VHCH1-Fchole_PGLALA-
CACCGGCTACTACATCCACTGGGTGAGGCAGGCCCCCGGCAAG DP47 VL
GGCCTGGAGTGGGTGGCCAGGGTGATCCCCAACGCCGGCGGCA
CCAGCTACAACCAGAAGTTCAAGGGCAGGTTCACCCTGAGCGT
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
AGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCA
TCTACTGGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGC
CAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGC
AAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGA
AGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGG
CGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAG
AGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCA
GCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCA
CAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAG
AGCTGCGACGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGG
TGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGG
CAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTACAGCTTCACC
GGCTACTACATCCACTGGGTGAGGCAGGCCCCCGGCAAGGGCC
TGGAGTGGGTGGCCAGGGTGATCCCCAACGCCGGCGGCACCAG
CTACAACCAGAAGTTCAAGGGCAGGTTCACCCTGAGCGTGGAC
AACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAGGG
CCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCATCTA
CTGGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGC
ACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGA
GCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGA
CTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCC
CTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCA
GCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAG
CAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT
GCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGGC
CGCCGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG
GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGG
TGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCC
AGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGC
TGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAA
GTGCAAGGTGAGCAACAAGGCCCTGGGCGCCCCCATCGAGAAG
ACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGT
GCACCCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGT
GAGCCTGAGCTGCGCCGTGAAGGGCTTCTACCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACA
AGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCT
GGTGAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGC
AACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACC
ACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCGGCGGCGG
CGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC
GGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGA
GCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAG
CCAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAG
CCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCA
GGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGG
CACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGAC
TTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCCCCCTGA
CCTTCGGCCAGGGCACCAAGGTGGAGATCAAG +CD40 LC see above 106 pETR15390
CD40 x DP47 (4 + 2) CD40
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG 117 VHCH1-CD40
GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACAGCTT VHCH1-
CACCGGCTACTACATCCACTGGGTGCGCCAGGCCCCTGGCAAG Fc_PGLALA-
GGACTGGAATGGGTGGCCAGAGTGATCCCCAATGCCGGCGGAA DP47 VLCH1
CCAGCTACAACCAGAAGTTCAAGGGCCGGTTCACCCTGAGCGT `EE`
GGACAACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTG
CGGGCCGAGGACACCGCCGTGTACTATTGTGCCCGCGAGGGCA
TCTATTGGTGGGGCCAGGGAACACTCGTGACCGTGTCCAGCGC
TTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGC
AAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGG
AGGACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG
CGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTCCAG
AGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACTGTGCCCA
GCAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGAACCA
CAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAACCCAAG
AGCTGCGACGGCGGAGGCGGATCTGGCGGCGGAGGATCCGAAG
TGCAGCTGGTGGAAAGTGGGGGAGGCCTGGTGCAGCCAGGGGG
AAGCCTGAGACTGTCTTGTGCCGCTTCCGGCTACTCTTTTACC
GGGTATTATATCCATTGGGTGCGGCAGGCTCCAGGGAAAGGCC
TGGAATGGGTGGCACGCGTGATCCCTAACGCAGGCGGCACCTC
TTATAATCAGAAGTTTAAAGGGCGCTTTACCCTGTCCGTGGAC
AATTCCAAGAATACTGCTTACCTGCAGATGAATTCCCTGCGCG
CCGAAGATACAGCTGTGTATTACTGCGCCAGAGAAGGGATCTA
TTGGTGGGGACAGGGCACCCTCGTGACAGTGTCATCCGCTAGC
ACCAAGGGACCTTCCGTGTTTCCCCTGGCTCCCAGCTCCAAGT
CTACCTCTGGGGGCACAGCTGCTCTGGGATGTCTGGTGGAAGA
TTATTTTCCTGAACCTGTGACCGTGTCATGGAACAGCGGAGCC
CTGACCTCCGGGGTGCACACATTCCCTGCTGTGCTGCAGTCCT
CCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCAG
CTCTCTGGGCACACAGACATATATCTGTAATGTGAATCACAAA
CCCTCTAATACCAAAGTGGATGAGAAAGTGGAACCTAAGTCCT
GCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGC
TGCTGGCGGCCCATCTGTGTTTCTGTTCCCCCCAAAGCCCAAG
GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGG
TGGTGGATGTGTCCCACGAGGACCCAGAAGTGAAGTTCAATTG
GTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCG
CGGGAAGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGC
TGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAA
GTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAGAAA
ACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAACCTCAGGTGT
ACACCCTGCCCCCAAGCAGGGACGAGCTGACCAAGAACCAGGT
GTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATC
GCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACA
AGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCT
GTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGC
AACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACC
ACTACACACAGAAGTCTCTGAGCCTGAGCCCTGGCGGAGGGGG
AGGATCTGGGGGAGGCGGAAGTGGGGGAGGGGGTTCCGGAGGC
GGAGGATCCGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGT
CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAG
TCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAA
CCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCA
GGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGG
GACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGAT
TTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTGA
CGTTCGGCCAGGGGACCAAAGTGGAAATCAAAAGCAGCGCTTC
CACCAAGGGCCCCTCAGTGTTCCCACTGGCACCATCCAGCAAG
TCCACAAGCGGAGGAACCGCCGCTCTGGGCTGTCTCGTGAAAG
ACTACTTTCCAGAGCCAGTGACCGTGTCCTGGAATAGTGGCGC
TCTGACTTCTGGCGTGCACACTTTCCCCGCAGTGCTGCAGAGT
TCTGGCCTGTACTCCCTGAGTAGCGTCGTGACAGTGCCCTCCT
CTAGCCTGGGCACTCAGACTTACATCTGCAATGTGAATCATAA
GCCTTCCAACACAAAAGTGGACAAAAAAGTGGAACCCAAATCT TGC +CD40 see above 110
LC; ,RK` pETR15391 +DP47VHCL
GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG 118 pETR15119
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTT
TAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAG
AGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTG
AGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCG
GATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAG
TGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCC
GACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGC
TGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGT
GGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTGACC
GAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCC
TGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGC
CTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAG TCTTTCAACCGGGGCGAGTGC
mu CD40-28H1 (4 + 1) CD40
GAAGTGCAGCTGGTGGAAAGCGACGGCGGACTGGTGCAGCCTG 119 VHCH1-CD40
GCAGATCTCTGAAGCTGCCTTGTGCCGCCAGCGGCTTCACCTT VHCH1-
CAGCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG FcKK_DAPG-
GGACTGGAATGGGTGGCCAGCATCAGCTACGACGGCAGCAGCA 28H1 VH
CCTACTACAGAGACAGCGTGAAGGGCAGATTCACCATCAGCAG pETR15732
AGACAACGCCAAGAGCACCCTGTACCTGCAGATGGACAGCCTG
AGAAGCGAGGACACCGCTACCTACTACTGCGGCAGACACAGCA
GCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTC
TAGCGCCAAGACCACACCCCCCAGCGTGTACCCTCTGGCTCCT
GGATCTGCCGCCCAGACCAACAGCATGGTCACACTGGGCTGCC
TGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAA
CAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTCCCTGCCGTG
CTGCAGAGCGACCTGTACACCCTGTCCTCCAGCGTGACCGTGC
CTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGC
CCACCCTGCCAGCTCCACCAAGGTGGACAAGAAAATCGTGCCC
CGGGACTGCGGAGGGGGCGGTTCCGGCGGAGGAGGATCCGAGG
TGCAGCTGGTGGAATCTGATGGGGGCCTGGTGCAGCCCGGAAG
AAGCCTGAAACTGCCCTGTGCTGCCTCTGGCTTCACATTCTCT
GATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCC
TGGAATGGGTGGCATCCATCTCTTACGACGGCTCCTCCACTTA
CTACAGGGACTCTGTGAAGGGCCGGTTCACAATCTCCCGGGAT
AACGCCAAGTCTACACTGTACCTGCAGATGGATTCCCTGCGCT
CCGAGGACACAGCCACATATTACTGTGGCAGGCACTCCTCCTA
CTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCCAGC
GCTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCCTGGAT
CTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCTGCCTGGT
GAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGC
GGCAGCCTGAGCAGCGGCGTGCACACCTTTCCAGCCGTGCTGC
AGAGCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAG
CAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCAC
CCTGCCAGCAGCACCAAGGTGGACAAGAAAATCGTGCCCCGGG
ACTGCGGCTGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTC
CAGCGTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTGACC
ATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCA
GCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTTGTGGACGA
CGTGGAGGTGCACACAGCCCAGACAAAGCCCCGGGAGGAACAG
ATCAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGC
ACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAA
CAGCGCCGCCTTCGGCGCCCCCATCGAGAAAACCATCAGCAAG
ACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACCATCCCCC
CACCCAAAAAACAGATGGCCAAGGACAAGGTGTCCCTGACCTG
CATGATCACCAACTTTTTCCCCGAGGACATCACCGTGGAGTGG
CAGTGGAATGGCCAGCCCGCCGAGAACTACAAGAACACCCAGC
CCATCATGAAGACCGACGGCAGCTACTTCGTGTACAGCAAGCT
GAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACC
TGTAGCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGA
AGTCCCTGAGCCACTCCCCCGGCGGCGGAGGCGGTTCCGGAGG
AGGAGGATCCGGAGGAGGGGGAAGTGGCGGCGGAGGATCTGAG
GTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTGGCG
GATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTC
CTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGC
CTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACT
ACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAA
CTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCC
GAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCA
ACTTCGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCAG C CD40
GAAGTGCAGCTGGTGGAAAGCGACGGCGGACTGGTGCAGCCTG 120 VHCH1-CD40
GCAGATCTCTGAAGCTGCCTTGTGCCGCCAGCGGCTTCACCTT VHCH1-
CAGCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG FcDD_DAPG-
GGACTGGAATGGGTGGCCAGCATCAGCTACGACGGCAGCAGCA 28H1 VL
CCTACTACAGAGACAGCGTGAAGGGCAGATTCACCATCAGCAG pETR15731
AGACAACGCCAAGAGCACCCTGTACCTGCAGATGGACAGCCTG
AGAAGCGAGGACACCGCTACCTACTACTGCGGCAGACACAGCA
GCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTC
TAGCGCCAAGACCACACCCCCCAGCGTGTACCCTCTGGCTCCT
GGATCTGCCGCCCAGACCAACAGCATGGTCACACTGGGCTGCC
TGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAA
CAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTCCCTGCCGTG
CTGCAGAGCGACCTGTACACCCTGTCCTCCAGCGTGACCGTGC
CTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGC
CCACCCTGCCAGCTCCACCAAGGTGGACAAGAAAATCGTGCCC
CGGGACTGCGGAGGGGGCGGTTCCGGCGGAGGAGGATCCGAGG
TGCAGCTGGTGGAATCTGATGGGGGCCTGGTGCAGCCCGGAAG
AAGCCTGAAACTGCCCTGTGCTGCCTCTGGCTTCACATTCTCT
GATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCC
TGGAATGGGTGGCATCCATCTCTTACGACGGCTCCTCCACTTA
CTACAGGGACTCTGTGAAGGGCCGGTTCACAATCTCCCGGGAT
AACGCCAAGTCTACACTGTACCTGCAGATGGATTCCCTGCGCT
CCGAGGACACAGCCACATATTACTGTGGCAGGCACTCCTCCTA
CTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCCAGC
GCTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCCTGGAT
CTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCTGCCTGGT
GAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGC
GGCAGCCTGAGCAGCGGCGTGCACACCTTTCCAGCCGTGCTGC
AGAGCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAG
CAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCAC
CCTGCCAGCAGCACCAAGGTGGACAAGAAAATCGTGCCCCGGG
ACTGCGGCTGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTC
CAGCGTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTGACC
ATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCA
GCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTTGTGGACGA
CGTGGAGGTGCACACAGCCCAGACAAAGCCCCGGGAGGAACAG
ATCAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGC
ACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAA
CAGCGCCGCCTTCGGCGCCCCCATCGAGAAAACCATCAGCAAG
ACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACCATCCCCC
CACCCAAAGAACAGATGGCCAAGGACAAGGTGTCCCTGACCTG
CATGATCACCAACTTTTTCCCCGAGGACATCACCGTGGAGTGG
CAGTGGAATGGCCAGCCCGCCGAGAACTACGACAACACCCAGC
CCATCATGGACACCGACGGCAGCTACTTCGTGTACAGCGACCT
GAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACC
TGTAGCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGA
AGTCCCTGAGCCACAGCCCAGGCGGCGGAGGCGGATCTGGCGG
AGGAGGTTCCGGAGGTGGCGGATCTGGGGGCGGTGGATCTGAG
ATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCTGAGCCCTG
GCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGAG
CCGGAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCC
CCCAGACTGCTGATCATCGGCGCCAGCACCCGGGCCACCGGCA
TCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCAC
CCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTAC
TACTGCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCCAGG GCACCAAGGTGGAAATCAAG mu
CD40 light GACACTGTACTGACCCAGTCTCCTGCTTTGGCTGTGTCTCCAG 121 chain
GAGAGAGGGTTACCATCTCCTGTAGGGCCAGTGACAGTGTCAG pETR13185
TACACTTATGCACTGGTACCAACAGAAACCAGGACAGCAACCC
AAACTCCTCATCTATCTAGCATCACACCTAGAATCTGGGGTCC
CTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT
CACCATTGATCCTGTGGAGGCTGATGACACTGCAACCTATTAC
TGTCAGCAGAGTTGGAATGATCCGTGGACGTTCGGTGGAGGCA
CCAAGCTGGAATTGAAACGTGCCGATGCTGCACCAACTGTATC
GATTTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCC
TCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCA
ATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGT
CCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTAC
AGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAAC
GACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAAC
TTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT mu CD40- 28H1 (4 + 2) CD40
GAAGTGCAGCTGGTGGAATCCGACGGCGGACTGGTGCAGCCTG 122 VHCH1-CD40
GCAGATCTCTGAAGCTGCCTTGTGCCGCCTCCGGCTTCACCTT VHCH1-
CTCCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG Fc_DAPG-
GGACTGGAATGGGTGGCCTCCATCTCCTACGACGGCTCCTCCA 28H1 VLCH1
CCTACTACCGGGACTCTGTGAAGGGCCGGTTCACCATCTCTCG pETR15744
GGACAACGCCAAGTCCACCCTGTACCTGCAGATGGACTCCCTG
CGGAGCGAGGACACCGCTACCTACTACTGCGGCAGACACTCCT
CCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTC
CTCTGCTAAGACCACCCCCCCCTCCGTGTACCCTCTGGCTCCT
GGATCTGCCGCCCAGACCAACTCCATGGTCACCCTGGGCTGTC
TGGTGGAAGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAA
CTCCGGCTCTCTGTCCTCTGGCGTGCACACCTTCCCTGCCGTG
CTGCAGTCCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGC
CTAGCAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGC
CCACCCTGCCAGCAGCACCAAGGTGGACGAGAAAATCGTGCCC
CGGGACTGCGGCGGTGGAGGTTCCGGAGGCGGCGGATCCGAGG
TGCAGCTGGTGGAAAGTGATGGGGGCCTGGTGCAGCCCGGAAG
AAGCCTGAAACTGCCCTGCGCCGCTTCTGGCTTTACCTTTAGC
GATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCC
TGGAATGGGTGGCATCTATCAGCTACGATGGCAGCAGCACCTA
CTATAGAGACAGCGTGAAGGGGAGATTCACCATCAGCAGAGAT
AACGCTAAGAGCACACTGTACCTGCAGATGGATAGCCTGAGAT
CCGAGGATACCGCCACATATTACTGTGGCCGGCACAGCAGCTA
CTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCTAGC
GCTAAGACTACCCCTCCTAGCGTGTACCCCCTGGCACCAGGTT
CCGCTGCTCAGACCAACAGCATGGTCACACTGGGATGCCTGGT
GGAAGGATATTTTCCTGAACCCGTGACAGTGACATGGAATAGC
GGCTCCCTGTCTAGCGGAGTGCATACCTTTCCAGCTGTGCTGC
AGAGCGATCTGTATACACTGAGCAGCTCTGTGACAGTGCCTTC
CAGCACCTGGCCCAGCCAGACAGTGACCTGTAATGTGGCTCAT
CCCGCCTCTAGCACCAAAGTGGATGAGAAAATCGTGCCCCGGG
ACTGCGGCTGCAAGCCCTGTATCTGTACCGTGCCCGAGGTGTC
CTCCGTGTTCATCTTCCCACCTAAGCCCAAGGACGTGCTGACA
ATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCT
CCAAGGACGATCCCGAGGTGCAGTTCAGTTGGTTCGTGGACGA
CGTGGAAGTGCACACAGCCCAGACAAAGCCCAGAGAGGAACAG
ATCAACTCCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGC
ACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAA
CTCCGCCGCCTTTGGCGCCCCTATCGAAAAGACCATCTCCAAG
ACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACAATCCCCC
CACCCAAAGAACAGATGGCCAAGGACAAGGTGTCCCTGACCTG
CATGATCACCAACTTTTTCCCAGAGGACATCACCGTGGAATGG
CAGTGGAACGGCCAGCCCGCCGAGAACTACAAGAACACCCAGC
CCATCATGGACACCGACGGCTCCTACTTCGTGTACTCCAAGCT
GAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACC
TGTTCCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGA
AGTCCCTGTCCCACTCCCCTGGAAAAGGCGGAGGCGGATCTGG
TGGCGGAGGATCTGGCGGTGGTGGTTCCGGAGGCGGTGGATCT
GAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCACTGTCTC
CAGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCTCAGTCCGT
GTCCCGGTCTTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAG
GCTCCCCGGCTGCTGATCATCGGAGCTTCTACCAGAGCCACCG
GCATCCCCGACAGATTCTCCGGCTCTGGCTCTGGCACCGACTT
CACCCTGACCATCTCTCGGCTGGAACCCGAGGACTTCGCCGTG
TACTACTGCCAGCAGGGCCAAGTGATCCCCCCCACCTTTGGCC
AGGGCACCAAGGTGGAAATCAAGTCCAGCGCTAAGACCACCCC
CCCCTCCGTGTATCCTCTGGCCCCTGGATCTGCCGCCCAGACC
AACTCCATGGTCACCCTGGGCTGCCTCGTGAAGGGCTACTTCC
CTGAGCCTGTGACCGTGACCTGGAACTCCGGCTCCCTGTCTAG
CGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCGACCTGTAC
ACCCTGAGCAGCTCCGTGACCGTGCCTTCCTCCACCTGGCCTT
CCCAGACCGTGACATGCAACGTGGCCCACCCTGCCAGCTCCAC
AAAGGTGGACAAGAAAATCGTGCCCCGGGACTGC 28H1 VHCL
GAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCCTG 123 (mu)
GCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTT pETR15650
CTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAA
GGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGT
ACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGA
CAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGG
GCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGG
GCAACTTCGACTACTGGGGACAGGGCACCCTGGTCACCGTGTC
CAGCGCTTCTGATGCCGCCCCTACCGTATCGATTTTCCCACCC
TCCAGCGAGCAGCTGACAAGCGGCGGAGCTAGCGTCGTGTGCT
TCCTGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAA
GATCGACGGCAGCGAGCGGCAGAACGGCGTGCTGAATAGCTGG
ACCGACCAGGACAGCAAGGACTCCACCTACAGCATGAGCAGCA
CCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTA
CACATGCGAGGCCACCCACAAGACCAGCACCAGCCCCATCGTG
AAGTCCTTCAACCGGAACGAGTGC mu CD40 light
GACACTGTACTGACCCAGTCTCCTGCTTTGGCTGTGTCTCCAG 124 chain; `RK`
GAGAGAGGGTTACCATCTCCTGTAGGGCCAGTGACAGTGTCAG pETR15649
TACACTTATGCACTGGTACCAACAGAAACCAGGACAGCAACCC
AAACTCCTCATCTATCTAGCATCACACCTAGAATCTGGGGTCC
CTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT
CACCATTGATCCTGTGGAGGCTGATGACACTGCAACCTATTAC
TGTCAGCAGAGTTGGAATGATCCGTGGACGTTCGGTGGAGGCA
CCAAGCTGGAATTGAAACGTGCCGATGCTGCACCAACTGTATC
GATTTTCCCACCATCCAGTCGGAAGTTAACATCTGGAGGTGCC
TCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCA
ATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGT
CCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTAC
AGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAAC
GACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAAC
TTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT mu CD40-DP47 (4 + 1) CD40
GAAGTGCAGCTGGTGGAAAGCGACGGCGGACTGGTGCAGCCTG 125 VHCH1-CD40
GCAGATCTCTGAAGCTGCCTTGTGCCGCCAGCGGCTTCACCTT VHCH1-
CAGCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG FcKK_DAPG-
GGACTGGAATGGGTGGCCAGCATCAGCTACGACGGCAGCAGCA DP47 VH
CCTACTACAGAGACAGCGTGAAGGGCAGATTCACCATCAGCAG pETR15734
AGACAACGCCAAGAGCACCCTGTACCTGCAGATGGACAGCCTG
AGAAGCGAGGACACCGCTACCTACTACTGCGGCAGACACAGCA
GCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTC
TAGCGCCAAGACCACACCCCCCAGCGTGTACCCTCTGGCTCCT
GGATCTGCCGCCCAGACCAACAGCATGGTCACACTGGGCTGCC
TGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAA
CAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTCCCTGCCGTG
CTGCAGAGCGACCTGTACACCCTGTCCTCCAGCGTGACCGTGC
CTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGC
CCACCCTGCCAGCTCCACCAAGGTGGACAAGAAAATCGTGCCC
CGGGACTGCGGAGGGGGCGGTTCCGGCGGAGGAGGATCCGAGG
TGCAGCTGGTGGAATCTGATGGGGGCCTGGTGCAGCCCGGAAG
AAGCCTGAAACTGCCCTGTGCTGCCTCTGGCTTCACATTCTCT
GATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCC
TGGAATGGGTGGCATCCATCTCTTACGACGGCTCCTCCACTTA
CTACAGGGACTCTGTGAAGGGCCGGTTCACAATCTCCCGGGAT
AACGCCAAGTCTACACTGTACCTGCAGATGGATTCCCTGCGCT
CCGAGGACACAGCCACATATTACTGTGGCAGGCACTCCTCCTA
CTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCCAGC
GCTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCCTGGAT
CTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCTGCCTGGT
GAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGC
GGCAGCCTGAGCAGCGGCGTGCACACCTTTCCAGCCGTGCTGC
AGAGCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAG
CAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCAC
CCTGCCAGCAGCACCAAGGTGGACAAGAAAATCGTGCCCCGGG
ACTGCGGCTGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTC
CAGCGTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTGACC
ATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCA
GCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTTGTGGACGA
CGTGGAGGTGCACACAGCCCAGACAAAGCCCCGGGAGGAACAG
ATCAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGC
ACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAA
CAGCGCCGCCTTCGGCGCCCCCATCGAGAAAACCATCAGCAAG
ACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACCATCCCCC
CACCCAAAAAACAGATGGCCAAGGACAAGGTGTCCCTGACCTG
CATGATCACCAACTTTTTCCCCGAGGACATCACCGTGGAGTGG
CAGTGGAATGGCCAGCCCGCCGAGAACTACAAGAACACCCAGC
CCATCATGAAGACCGACGGCAGCTACTTCGTGTACAGCAAGCT
GAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACC
TGTAGCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGA
AGTCCCTGAGCCACTCCCCCGGCGGCGGAGGCGGTTCCGGAGG
AGGAGGATCCGGAGGAGGGGGAAGTGGCGGCGGAGGATCTGAG
GTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGG
GGTCCCTGAGACTCTCCTGTGCAGCCAGCGGATTCACCTTTAG
CAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG
CTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACAT
ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGA
GCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGAT
TTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGC CD40
GAAGTGCAGCTGGTGGAAAGCGACGGCGGACTGGTGCAGCCTG 126 VHCH1-CD40
GCAGATCTCTGAAGCTGCCTTGTGCCGCCAGCGGCTTCACCTT VHCH1-
CAGCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG FcDD_DAPG-
GGACTGGAATGGGTGGCCAGCATCAGCTACGACGGCAGCAGCA DP47 VL
CCTACTACAGAGACAGCGTGAAGGGCAGATTCACCATCAGCAG pETR15733
AGACAACGCCAAGAGCACCCTGTACCTGCAGATGGACAGCCTG
AGAAGCGAGGACACCGCTACCTACTACTGCGGCAGACACAGCA
GCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTC
TAGCGCCAAGACCACACCCCCCAGCGTGTACCCTCTGGCTCCT
GGATCTGCCGCCCAGACCAACAGCATGGTCACACTGGGCTGCC
TGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAA
CAGCGGCTCTCTGTCTAGCGGCGTGCACACCTTCCCTGCCGTG
CTGCAGAGCGACCTGTACACCCTGTCCTCCAGCGTGACCGTGC
CTTCCTCCACCTGGCCTTCCCAGACCGTGACATGCAACGTGGC
CCACCCTGCCAGCTCCACCAAGGTGGACAAGAAAATCGTGCCC
CGGGACTGCGGAGGGGGCGGTTCCGGCGGAGGAGGATCCGAGG
TGCAGCTGGTGGAATCTGATGGGGGCCTGGTGCAGCCCGGAAG
AAGCCTGAAACTGCCCTGTGCTGCCTCTGGCTTCACATTCTCT
GATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCC
TGGAATGGGTGGCATCCATCTCTTACGACGGCTCCTCCACTTA
CTACAGGGACTCTGTGAAGGGCCGGTTCACAATCTCCCGGGAT
AACGCCAAGTCTACACTGTACCTGCAGATGGATTCCCTGCGCT
CCGAGGACACAGCCACATATTACTGTGGCAGGCACTCCTCCTA
CTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCCAGC
GCTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCCTGGAT
CTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCTGCCTGGT
GAAGGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAACAGC
GGCAGCCTGAGCAGCGGCGTGCACACCTTTCCAGCCGTGCTGC
AGAGCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGCCTAG
CAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGCCCAC
CCTGCCAGCAGCACCAAGGTGGACAAGAAAATCGTGCCCCGGG
ACTGCGGCTGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTC
CAGCGTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTGACC
ATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCA
GCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTTGTGGACGA
CGTGGAGGTGCACACAGCCCAGACAAAGCCCCGGGAGGAACAG
ATCAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGC
ACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAA
CAGCGCCGCCTTCGGCGCCCCCATCGAGAAAACCATCAGCAAG
ACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACCATCCCCC
CACCCAAAGAACAGATGGCCAAGGACAAGGTGTCCCTGACCTG
CATGATCACCAACTTTTTCCCCGAGGACATCACCGTGGAGTGG
CAGTGGAATGGCCAGCCCGCCGAGAACTACGACAACACCCAGC
CCATCATGGACACCGACGGCAGCTACTTCGTGTACAGCGACCT
GAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACC
TGTAGCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGA
AGTCCCTGAGCCACAGCCCAGGCGGCGGAGGCGGATCTGGCGG
AGGAGGTTCCGGAGGCGGCGGAAGCGGAGGGGGAGGCTCTGAA
ATTGTGCTGACCCAGAGCCCCGGCACCCTGTCACTGTCTCCAG
GCGAAAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGTC
CAGCTCTTACCTGGCCTGGTATCAGCAGAAGCCCGGACAGGCC
CCCAGACTGCTGATCTACGGCGCCTCTTCTAGAGCCACCGGCA
TCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCAC
CCTGACAATCAGCAGACTGGAACCCGAGGACTTTGCCGTGTAT
TACTGCCAGCAGTACGGCAGCAGCCCCCTGACCTTTGGCCAGG GCACCAAGGTGGAAATCAAA
+mu CD40 see above 121 light chain mu CD40 x DP47 (4 + 2) CD40
GAAGTGCAGCTGGTGGAATCCGACGGCGGACTGGTGCAGCCTG 127 VHCH1-CD40
GCAGATCTCTGAAGCTGCCTTGTGCCGCCTCCGGCTTCACCTT VHCH1-
CTCCGACTACTACATGGCCTGGGTGCGACAGGCCCCTACCAAG Fc_DAPG-
GGACTGGAATGGGTGGCCTCCATCTCCTACGACGGCTCCTCCA 28H1 VLCH1
CCTACTACCGGGACTCTGTGAAGGGCCGGTTCACCATCTCTCG `EE`
GGACAACGCCAAGTCCACCCTGTACCTGCAGATGGACTCCCTG pETR15748
CGGAGCGAGGACACCGCTACCTACTACTGCGGCAGACACTCCT
CCTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCGTGTC
CTCTGCTAAGACCACCCCCCCCTCCGTGTACCCTCTGGCTCCT
GGATCTGCCGCCCAGACCAACTCCATGGTCACCCTGGGCTGTC
TGGTGGAAGGCTACTTCCCCGAGCCTGTGACCGTGACCTGGAA
CTCCGGCTCTCTGTCCTCTGGCGTGCACACCTTCCCTGCCGTG
CTGCAGTCCGACCTGTACACCCTGAGCAGCTCCGTGACCGTGC
CTAGCAGCACCTGGCCCAGCCAGACAGTGACCTGCAACGTGGC
CCACCCTGCCAGCAGCACCAAGGTGGACGAGAAAATCGTGCCC
CGGGACTGCGGCGGTGGAGGTTCCGGAGGCGGCGGATCCGAGG
TGCAGCTGGTGGAAAGTGATGGGGGCCTGGTGCAGCCCGGAAG
AAGCCTGAAACTGCCCTGCGCCGCTTCTGGCTTTACCTTTAGC
GATTACTATATGGCTTGGGTGCGCCAGGCTCCAACAAAAGGCC
TGGAATGGGTGGCATCTATCAGCTACGATGGCAGCAGCACCTA
CTATAGAGACAGCGTGAAGGGGAGATTCACCATCAGCAGAGAT
AACGCTAAGAGCACACTGTACCTGCAGATGGATAGCCTGAGAT
CCGAGGATACCGCCACATATTACTGTGGCCGGCACAGCAGCTA
CTTTGATTATTGGGGACAGGGCGTGATGGTCACAGTGTCTAGC
GCTAAGACTACCCCTCCTAGCGTGTACCCCCTGGCACCAGGTT
CCGCTGCTCAGACCAACAGCATGGTCACACTGGGATGCCTGGT
GGAAGGATATTTTCCTGAACCCGTGACAGTGACATGGAATAGC
GGCTCCCTGTCTAGCGGAGTGCATACCTTTCCAGCTGTGCTGC
AGAGCGATCTGTATACACTGAGCAGCTCTGTGACAGTGCCTTC
CAGCACCTGGCCCAGCCAGACAGTGACCTGTAATGTGGCTCAT
CCCGCCTCTAGCACCAAAGTGGATGAGAAAATCGTGCCCCGGG
ACTGCGGCTGCAAGCCCTGTATCTGTACCGTGCCCGAGGTGTC
CTCCGTGTTCATCTTCCCACCTAAGCCCAAGGACGTGCTGACA
ATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCCATCT
CCAAGGACGATCCCGAGGTGCAGTTCAGTTGGTTCGTGGACGA
CGTGGAAGTGCACACAGCCCAGACAAAGCCCAGAGAGGAACAG
ATCAACTCCACCTTCAGAAGCGTGTCCGAGCTGCCCATCATGC
ACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCAGAGTGAA
CTCCGCCGCCTTTGGCGCCCCTATCGAAAAGACCATCTCCAAG
ACCAAGGGCAGACCCAAGGCCCCCCAGGTGTACACAATCCCCC
CACCCAAAGAACAGATGGCCAAGGACAAGGTGTCCCTGACCTG
CATGATCACCAACTTTTTCCCAGAGGACATCACCGTGGAATGG
CAGTGGAACGGCCAGCCCGCCGAGAACTACAAGAACACCCAGC
CCATCATGGACACCGACGGCTCCTACTTCGTGTACTCCAAGCT
GAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCACC
TGTTCCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGA
AGTCCCTGTCCCACTCCCCTGGAAAAGGCGGAGGCGGATCTGG
TGGCGGAGGATCTGGCGGTGGTGGTTCCGGAGGCGGAGGATCC
GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTC
CAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGT
TAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAG
GCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTG
GCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTT
CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTG
TATTACTGTCAGCAGTATGGTAGCTCACCGCTGACGTTCGGCC
AGGGGACCAAAGTGGAAATCAAAAGCAGCGCTAAGACCACCCC
CCCCTCCGTGTATCCTCTGGCCCCTGGATCTGCCGCCCAGACC
AACTCCATGGTCACCCTGGGCTGCCTCGTGAAGGGCTACTTCC
CTGAGCCTGTGACCGTGACCTGGAACTCCGGCTCCCTGTCTAG
CGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCGACCTGTAC
ACCCTGAGCAGCTCCGTGACCGTGCCTTCCTCCACCTGGCCTT
CCCAGACCGTGACATGCAACGTGGCCCACCCTGCCAGCTCCAC
AAAGGTGGACAAGAAAATCGTGCCCCGGGACTGC +mu CD40 see above 124 light
chain; ,RK` DP47 VHCL GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG
128 (mu) GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTT pETR15652
TAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAG
AGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTG
AGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCG
GATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAG
CGCTTCTGATGCCGCCCCTACCGTATCGATTTTCCCACCCTCC
AGCGAGCAGCTGACAAGCGGCGGAGCTAGCGTCGTGTGCTTCC
TGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGAT
CGACGGCAGCGAGCGGCAGAACGGCGTGCTGAATAGCTGGACC
GACCAGGACAGCAAGGACTCCACCTACAGCATGAGCAGCACCC
TGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACAC
ATGCGAGGCCACCCACAAGACCAGCACCAGCCCCATCGTGAAG
TCCTTCAACCGGAACGAGTGC
1.2 Production of Bispecific Antigen Binding Molecules Targeting
CD40 and Fibroblast Activation Protein (FAP)
[0726] The molecules were produced by co-transfecting either
HEK293-EBNA cells growing in suspension with the mammalian
expression vectors using polyethylenimine (PEI) or co-transfecting
CHO K1 cells growing in suspension with the mammalian expression
using eviFECT. The cells were transfected with the corresponding
expression vectors.
[0727] For production in HEK293 EBNA cells HEK293 EBNA cells were
cultivated in suspension serum free in Excell culture medium
containing 6 mM L-glutamine and 250 mg/L G418. For the production
in 600 mL tubespin flasks (max. working volume 400 mL) 600 million
HEK293 EBNA cells were seeded 24 hours before transfection. For
transfection 800 million cells were centrifuged for 5 min at
210.times.g and supernatant was replaced by 20 mL pre-warmed CD CHO
medium. Expression vectors were mixed in 20 mL CD CHO medium to a
final amount of 400 .mu.g DNA. After addition of 1080 .mu.g PEI
solution (2.7 .mu.g/mL) the mixture was vortexed for 15 s and
subsequently incubated for 10 min at room temperature. Afterwards
cells were mixed with the DNA/PEI solution, transferred to a 600 mL
tubespin flask and incubated for 3 hours at 37.degree. C. in an
incubator with a 5% CO2 atmosphere. After incubation, 360 mL Excell
medium, containing 6 mM L-glutamine, 5 g/L Pepsoy and 1.25 mM VPA,
was added and cells were cultivated for 24 hours. One day after
transfection 12% Feed 7 and 3 g/1 Glucose were added. After 7 days
the cultivation supernatant was collected for purification by
centrifugation for 60 min at 2500.times.g (Sigma 8K centrifuge).
The solution was sterile filtered (0.22 .mu.m filter) and sodium
azide was added to a final concentration of 0.01% w/v and kept at
4.degree. C.
[0728] For production in HEK293 EBNA cells HEK293 EBNA in
suspension-adapted CHO K1 cells (adapted to serum-free growth in
suspension culture) the cells were grown in eviGrow medium (evitria
AG, Switzerland), a chemically defined, animal-component free,
serum-free medium and transfected with eviFect (evitria AG,
Switzerland). After transfection the cells were kept in eviMake
(evitria AG, Switzerland), a chemically defined, animal-component
free, serum-free medium, at 37.degree. C. and 5% CO.sub.2 for 7
days.
[0729] After 7 days the cultivation supernatant was collected for
purification by centrifugation for 45 min at maximum speed in a
Rotanta 460 RC. The solution was sterile filtered (0.22 .mu.m
filter) and kept at 4.degree. C. The concentration of the molecules
in the culture medium was either determined by Protein A-HPLC or
Protein A-Bio-Layer Interferometry (BLI).
[0730] The secreted protein was purified from cell culture
supernatants by affinity chromatography using Protein A affinity
chromatography, followed by a size exclusion chromatographic step.
For affinity chromatography supernatant was loaded on a HiTrap
MabSelect SuRe column (Column Volume (CV)=5 mL, GE Healthcare)
equilibrated with 25 ml 20 mM sodium phosphate, 20 mM sodium
citrate, pH 7.5. Unbound protein was removed by washing with at
least 10 CV 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5
and target protein was eluted in 6 CV 20 mM sodium citrate, 100 mM
sodium chloride, 100 mM glycine, pH 3.0. Protein solution was
neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8.0. The
target protein was concentrated and filtrated prior loading on a
HiLoad XK16/60 Superdex 200 column (GE Healthcare) equilibrated
with 20 mM histidine, 140 mM sodium chloride, pH 6.0, 0.01%
Tween20. The protein concentration of purified protein sample was
determined by measuring the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence.
[0731] Purity and molecular weight of the molecule after the final
purification step were analyzed by CE-SDS analyses in the presence
and absence of a reducing agent. The Caliper LabChip GXII system
(Caliper Lifescience) was used according to the manufacturer's
instruction.
[0732] The aggregate content of the molecule was analyzed using a
TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25
mM potassium phosphate, 125 mM sodium chloride, 200 mM L-arginine
monohydrocloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at
25.degree. C.
TABLE-US-00008 TABLE 5 Production yield and quality of bispecific
CD40 antigen binding molecules Concen- Yield tration Monomer HMW
LMW Construct [mg/L] [mg/ml] [%] [%] [%] huCD40-28H1; 4 + 1 48.2
8.9 97.8 1.2 1.0 huCD40-28H1; 4 + 2 48.1 9.2 99.7 0.3 --
huCD40-28H1; 2 + 1 10.1 2.0 99.5 0.5 -- huCD40-28H1; 2 + 2 26.7 2.0
100.0 -- -- huCD40-DP47; 4 + 1 15.5 2.3 98.7 1.3 -- huCD40-DP47; 4
+ 2 33.8 3.6 96.8 3.2 -- huCD40 IgG 190.3 11.8 99.8 0.2 --
muCD40-28H1; 4 + 1 3.4 0.5 92.4 7.6 -- muCD40-28H1; 4 + 2 3.9 1.6
98.9 0.4 0.7 muCD40 IgG 19.8 5.0 97.8 -- 2.2 muCD40-DP47; 4 + 1 2.2
0.3 90.6 9.4 -- muCD40-DP47; 4 + 2 1.5 0.6 86.5 7.8 5.7
1.3 Generation of Further Bispecific Antigen Binding Molecules
Targeting CD40 and Fibroblast Activation Protein (FAP)
[0733] In analogy to Example 1.1, different types of constructs of
bispecific CD40-FAP antibodies have been prepared. For example, a
bispecific antibody consisting of one CD40 binding moiety combined
with one FAP binding moiety was prepared (FIG. 15A). Because the
CrossMab technology as described in WO 2010/145792 A1 was used to
ensure correct light chain pairing, the format is called 1+1
crossmab. Another 2+1 format called "head-to-tail" was prepared
wherein a CD40 binding Fab is fused to the N-terminus of a FAP
binding Fab (FIG. 15B) and a further 2+1 format consisting of two
CD40 binding moieties combined with either one FAP binding crossfab
at the C-terminus of an Fc (FIG. 1E) was produced. Furthermore, a
4+1 format consisting of four CD40 binding moieties combined with
one FAP binding crossfab at the C-terminus of an Fc (FIG. 15C) was
prepared. In all these constructs, the variable heavy and light
chain domains of the anti CD40 binder correspond to the CD40 binder
as described in WO 2006/128103 (SEQ ID NO:10 and SEQ ID NO:16 of
said document). The generation and preparation of FAP binder 28H1
is described in WO 2012/020006 A2, which is incorporated herein by
reference. To generate the 1+1, 2+1 and 4+1 molecules the
knob-into-hole technology was used to achieve heterodimerization.
The S354C/T366W mutations have been introduced in the first heavy
chain HC1 (Fc knob heavy chain) and the Y349C/T366S/L368A/Y407V
mutations are introduced in the second heavy chain HC2 (Fc hole
heavy chain). Furthermore, the CrossMab technology as described in
WO 2010/145792 A1 ensures correct light chain pairing. Independent
of the bispecific format, in all cases an effector silent Fc
(P329G; L234, 234A) has been used to abrogate binding to Fc.gamma.
receptors according to the method described in WO 2012/130831 Al.
Amino acid Sequences of the bispecific molecules are shown in Table
6.
[0734] All genes are transiently expressed under control of a
chimeric MPSV promoter consisting of the MPSV core promoter
combined with the CMV promoter enhancer fragment. The expression
vector also contains the oriP region for episomal replication in
EBNA (Epstein Barr Virus Nuclear Antigen) containing host
cells.
TABLE-US-00009 TABLE 6 Amino acid sequences of the bispecific
antigen binding molecules Seq ID Construct Sequence No P1AE0192
CD40 x FAP (28H1) (1 + 1) crossmab 28H1 light chain
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCL
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC 28H1
EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQ 163
(VLCH1)_FCknob_PGLALA APRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV
YYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTL
PPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG CD40 (VHCH1
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 164
charged)_Fchole_PGLALA GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NRFTQKSLSLSPG CD40
light chain DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 165
(charged) KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0408 CD40 x
FAP (28H1) (2 + 1) head to tail 28H1 light chain
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCL
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC CD40 light
chain DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 165 (charged)
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (VHCH1
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 166 charged)_28H1
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL (VLCH1)_FCknob_PGLALA
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVS
RSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFT
LTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG
CD40 (VHCH1 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 164
charged)_Fchole_PGLALA GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NRFTQKSLSLSPGK CD40 x
FAP (28H1) (2 + 1) C-terminal crossfab fusion 28H1 light chain
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCL
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC CD40 light
chain DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 165 (charged)
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (VHCH1
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 167
charged)_Fcknob_PGLALA_28H1
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL (VLCH1)
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGT
LSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGAS
TRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIP
PTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSC CD40 (VHCH1
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 168
charged)_Fchole_PGLALA GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0637
CD40 x FAP (28H1) (4 + 1) C-terminal crossfab fusion 28H1 light
chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCL
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC CD40 light
chain DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 165 (charged)
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (VHCH1
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 169 charged_CD40
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL (VHCH1
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS
charged)-Fcknob_PGLALA_28H1
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ (VLCH1)
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQK
PGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPED
FAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C CD40 (VHCH1
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 170 charged_CD40
GLEWVARVIPNAGGTSYNQKFKGRFTLSVDNSKNTAYLQMNSL (VHCH1
RAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGPSVFPLAPSS charged)-Fchole_PGLALA
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVARVIPNAGGTSYNQKFKGRFTLSVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPG
[0735] Expression of the Bispecific Antibodies
[0736] Antibodies were expressed by transient transfection of HEK
cells grown in suspension with expression vectors encoding the 4
different peptide chains. Transfection into HEK293-F cells
(Invitrogen) was performed according to the cell supplier's
instructions using Maxiprep (Qiagen) preparations of the antibody
vectors, F17 medium (Invitrogen, USA), Peipro (Polyscience Europe
GmbH) and an initial cell density of 1-2 million viable cells/ml in
serum free FreeStyle 293 expression medium (Invitrogen). Cell
culture supernatants were harvested after 7 days of cultivation in
shake flasks or stirred fermenters by centrifugation at 14000 g for
30 minutes and filtered through a 0.22 .mu.m filter.
[0737] Purification of the Bispecific Antibodies
[0738] Antibodies were purified from cell culture supernatants by
affinity chromatography using MabSelectSure-Sepharose.TM. (GE
Healthcare, Sweden) chromatography. Briefly, sterile filtered cell
culture supernatants were captured on a MabSelect SuRe resin
equilibrated with PBS buffer (10 mM Na.sub.2HPO.sub.4, 1 mM
KH.sub.2PO.sub.4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with
equilibration buffer and eluted with 25 mM cirate, pH 3.0. After
neutralization with 1 M Tris pH 9.0, aggregated protein was
separated from monomeric antibody species by size exclusion
chromatography (Superdex 200, GE Healthcare) in 20 mM histidine,
140 mM NaCl, pH 6.0. Monomeric protein fractions were pooled,
concentrated if required using e.g. a MILLIPORE Amicon Ultra (30KD
MWCO) centrifugal concentrator and stored at -80.degree. C. Sample
aliquots were used for subsequent analytical characterization e.g.
by CE-SDS, size exclusion chromatography, mass spectrometry and
endotoxin determination.
1.4 Characterization of Bispecific Constructs Targeting CD40 and
FAP
1.4.1 Binding to Human or Mouse FAP-Expressing Murine Fibroblast
Cells
[0739] The binding to cell surface FAP was tested using human
fibroblast activating protein (huFAP) expressing cells
NIH/3T3-huFAP clone 19 or mouse fibroblast activating protein
(mFAP) expressing cells NIH/3T3-mFAP clone 26. NIH/3T3-huFAP clone
19 and NIH/3T3-mFAP clone 26 were generated by the transfection of
the mouse embryonic fibroblast NIH/3T3 cell line (ATCC CRL-1658)
with the expression vector pETR4921 to express under 1.5 .mu.g/mL
Puromycin selection huFAP or mFAP, respectively. NIH/3T3 wildtype
(wt) cells that were not transfected with FAP and that do not
express FAP were used as a negative control.
[0740] NIH/3T3-huFAP, NIH/3T3-mFAP or NIH/3T3-wt cells were
cultured with 1.times. Dulbecco's Modified Eagle's Medium (DMEM)
(gibco, Cat. No. 42430-025) supplemented with 10% Fetal Bovine
Serum (FBS) (life technologies, Cat. No. 16140, Lot No. 1797306A).
For the NIH/3T3-huFAP and NIH/3T3-mFAP cells 1.5 .mu.g/mL Puromycin
(gibco, Cat. No. A11138-03) was added to the medium for selection
of FAP-expressing cells. NIH/3T3 cells were removed from the plate
by using enzyme-free Cell Dissociation Buffer (gibco, Cat. No.
13151014). 0.3.times.10.sup.5 NIH/3T3-huFAP clone 19, NIH/3T3-mFAP
clone 26 or NIH/3T3-wt were added in 200 .mu.l of 1.times. DMEM 10%
FBS to each well of a round-bottom 96-well plate (greiner bio-one,
cellstar, Cat. No. 650185). Plates were centrifuged 5 minutes at
1700 rpm and supernatants were flicked off. Cells were washed once
with 200 .mu.L of 4.degree. C. cold FACS buffer (eBioscience, Cat.
No. 00-4222-26). All samples were resuspended in 50 .mu.L/well of
4.degree. C. cold FACS buffer containing the bispecific antigen
binding molecules (primary antibody) or isotype control antibody
DP47 at the indicated range of concentrations (in duplicates) and
incubated for 120 minutes at 4 .degree. C. Afterwards the cells
were washed three times with 200 .mu.L 4.degree. C. cold FACS
buffer. Cells were further stained with 25 .mu.L/well of 4 .degree.
C. cold secondary antibody solution (1:50 dilution of secondary
antibody) containing R-Phycoerythrin (PE) conjugated AffiniPure
F(ab').sub.2 Fragment Goat Anti-Human IgG, Fc.gamma. Fragment
Specific (Jackson ImmunoResearch, Cat. No. 109-116-098) and
incubated for 60 minutes at 4 .degree. C. in the dark. Cells were
washed with 200 .mu.l FACS buffer and resuspended in 85 .mu.L/well
FACS-buffer containing 0.2 .mu.g/mL DAPI (Roche, Cat. No.
10236276001) and acquired the same day using 5-laser LSR-Fortessa
(BD Bioscience with DIVA software). Data analysis was performed
using the FlowJo version 10 software (FlowJo LLC).
[0741] As shown in FIG. 2A, FIG. 2B and FIG. 17, the bispecific
antibodies monovalent or bivalent for FAP bind to human and mouse
FAP-expressing target cells. Therefore, only FAP-targeted anti-CD40
antigen binding molecules show direct tumor-targeting properties.
The bivalent FAP constructs with C-terminal FAP binding domains
bind stronger than the monovalent construct with C-terminal FAP
binding domain explained by a gain of avidity in the bivalent
relative to the monovalent FAP format. The strongest FAP binding
was observed for the 1+1 format (P1AE0192). No binding of the
FAP-targeted antibodies to the NIH/3T3-wt cells was detected. The
EC.sub.50 values as measured for different bispecific antibodies
are shown in Table 7 below.
TABLE-US-00010 TABLE 7 Human FAP binding characterization of 28H1
in different bispecific antibody formats Molecule EC.sub.50 [nM]
P1AD4470 CD40 IgG1 PGLALA n/a P1AE0637 CD40 .times. FAP 4 + 1 with
C- 10.46 terminal crossfab P1AD4453 CD40 .times. FAP 4 + 1 47.14
P1AD4574 CD40 .times. DP47 4 + 1 n/a P1AD4455 CD40 .times. FAP 4 +
2 3.64 P1AD4465 CD40 .times. DP47 4 + 2 n/a P1AA9641 CD40 .times.
FAP 2 + 1 32.96 P1AE0408 CD40 .times. FAP 2 + 1 head-to-tail 7.99
P1AA9663 CD40 .times. FAP 2 + 2 3.65 P1AE0192 CD40 .times. FAP 1 +
1 1.15 P1AE0889 CD40 (VH2a/VF2a) .times. FAP 24.26 4 + 1 with
C-terminal crossfab P1AE0890 CD40 (VH2a/VF2a) .times. FAP 30.42 4 +
1 with C-terminal crossfab
1.4.2 Binding to Human CD40-Expressing Daudi Cells
[0742] The binding to cell surface CD40 was tested using Daudi
cells, a human B lymphoblast cell line with high expression levels
of human CD40 (ATCC CCL-213). Daudi cells were cultured with
1.times. Dulbecco's Modified Eagle's Medium (DMEM) (gibco, Cat. No.
42430-025) supplemented with 10% Fetal Bovine Serum (FBS) (life
technologies, Cat. No. 16140, Lot No. 1797306A). 0.3.times.10.sup.5
Daudi cells were added in 200 .mu.l of 1.times. DMEM with 10% FBS
to each well of a round-bottom 96-well plate (greiner bio-one,
cellstar, Cat. No. 650185). Plates were centrifuged 5 minutes at
1700 rpm and supernatants were flicked off. Cells were washed once
with 200 .mu.L of 4.degree. C. cold FACS buffer (eBioscience, Cat.
No. 00-4222-26). All samples were resuspended in 50 .mu.L/well of
4.degree. C. cold FACS buffer containing the bispecific antigen
binding molecules (primary antibody) or isotype control antibody
DP47 at the indicated range of concentrations (in duplicates) and
incubated for 120 minutes at 4 .degree. C. Afterwards the cells
were washed three times with 200 .mu.L 4.degree. C. cold FACS
buffer. Cells were further stained with 25 .mu.L/well of 4.degree.
C. cold secondary antibody solution (1:50 dilution of secondary
antibody) containing R-Phycoerythrin (PE) conjugated AffiniPure
F(ab').sub.2 Fragment Goat Anti-Human IgG, Fc.gamma. Fragment
Specific (Jackson ImmunoResearch, Cat. No. 109-116-098) and
incubated for 60 minutes at 4.degree. C. in the dark. Cells were
washed with 200 .mu.l FACS buffer and resuspended in 85 .mu.L/well
FACS-buffer containing 0.2 .mu.g/mL DAPI (Roche, Cat. No.
10236276001) and acquired the same day using a 5-laser LSR-Fortessa
(BD Bioscience with DIVA software). Data analysis was performed
using the FlowJo version 10 software (FlowJo LLC).
[0743] As shown in FIG. 18, all depicted clones bind to CD40 but
vary in their binding strength (EC.sub.50 values as well as signal
strength) to CD40-positive Daudi cells. Bivalent anti-CD40
antibodies show higher EC.sub.50 levels and reach higher binding
plateaus compared to tetravalent anti-CD40 antibodies explained by
more occupied CD40 binding sites per antibody and a gain of avidity
of the tetravalent relative to the bivalent CD40 formats. The
highest EC.sub.50 value combined with the lowest binding plateau
was observed for the 1+1 format (P1AE0192). No binding of the
negative control antibody to Daudi cells was detected. The
EC.sub.50 values as measured for different bispecific antibodies
are shown in Table 8 below.
TABLE-US-00011 TABLE 8 Human CD40 binding characterization of CD40
antibodies in different bispecific antibody formats Molecule
EC.sub.50 [nM] P1AD4470 CD40 IgG1 PGLALA 0.104 P1AE0637 CD40
.times. FAP 4 + 1 with C- 0.015 terminal crossfab P1AD4453 CD40
.times. FAP 4 + 1 0.027 P1AD4574 CD40 .times. DP47 4 + 1 0.031
P1AD4455 CD40 .times. FAP 4 + 2 0.030 P1AD4465 CD40 .times. DP47 4
+ 2 0.036 P1AA9641 CD40 .times. FAP 2 + 1 0.079 P1AE0408 CD40
.times. FAP 2 + 1 head-to-tail 0.044 P1AA9663 CD40 .times. FAP 2 +
2 0.096 P1AE0192 CD40 .times. FAP 1 + 1 21.628 P1AE0889 CD40
(VH2a/VF2a) .times. FAP 0.046 4 + 1 with C-terminal crossfab
P1AE0890 CD40 (VH2a/VL2a) .times. FAP 0.048 4 + 1 with C-terminal
crossfab
Example 2
Functional Properties of FAP-Targeted Anti-Human CD40 Binding
Molecules
2.1 CD40-Mediated Activation of Antigen Presenting Cells (APCs) by
FAP-Targeted Anti-Human CD40 Binding Molecules
[0744] Ligation of CD40 induces B cell and dendritic cell (DC)
maturation as well as activation and promotes survival of these
cell types. Upon CD40 signaling cytokine production and
costimulatory molecule expression on the surface of B cells and DCs
is increased (S. Quezada et al., Annu RevImmunol. 2004, 22,
307-328; S. Danese et al., Gut. 2004, 53, 1035-1043; G. Bishop et
al., Adv Exp Med Biol. 2007, 597, 131-151).
[0745] In order to test the agonistic properties and the FAP
specificity of the different FAP-dependent anti-CD40 antibodies,
APCs obtained from human buffy coats were incubated with the
FAP-dependent agonistic anti-human CD40 antibodies and either
FAP-coated beads or human FAP expressing NIH/3T3 cells. APC
activation was measured by FACS and supernatant of the cells was
analyzed for cytokines by enzyme-linked immunosorbent assay
(ELISA).
2.1.1 Activation of Human B Cells by FAP-Targeted Anti-Human CD40
Binding Molecules using NIH/3T3-huFAP Cells as Source of
Antigen
[0746] NIH/3T3 cells expressing FAP were used as source of antigen
for the bispecific antigen binding molecules. The NIH/3T3-huFAP
cells were generated by transfection of mouse embryonic fibroblast
NIH/3T3 cells (ATCC CRL-1658) with the expression pETR4921 plasmid
encoding human FAP under a CMV promoter. The NIH/3T3-huFAP cells or
NIH/3T3-wt cells were irradiated with 27 Gy using a RS 2000
irradiator (Rad Source Technologies to prevent the cells from
proliferating. 0.2.times.10.sup.5 irradiated NIH/3T3 cells in 100
.mu.l of R10 medium consisting of Roswell Park Memorial Institute
medium (RPMI) 1640 (gibco, Cat. No. 31870-025) supplied with 10%
FBS, 1% (v/v) Penicillin Streptomycin (gibco, Cat. No. 15070-063),
1% (v/v) L-Glutamine (gibco, Cat. No.25030-024), 1% (v/v)
Sodium-Pyruvate (gibco, Cat. No. 11360-039), 1% (v/v) MEM
non-essential amino acids (gibco, Cat. No. 11140-035) and 50 .mu.M
(.beta.-Mercaptoethanol (gibco, Cat. No. 31350-010) were seeded per
well of a 96-well flat-bottom plate (TPP, Cat. No. 92696).
[0747] On the next day a buffy coat was obtained from the Stiftung
Ziircher Blutspendedienst SRK. In order to isolate peripheral blood
mononuclear cells (PBMCs), 50 mL of buffy coat were diluted in the
same volume of PBS (gibco, Cat. No. 10010023). 50 mL polypropylene
centrifuge tubes (TPP, Cat. No. 91050) were supplied with 15 mL of
Lymphoprep.TM. (STEMCELL Technologies, Cat. No. 07851) and 25 mL of
the buffy coat/PBS solution per tube were carefully layered above
the Lymphorep.TM.. The tubes were centrifuged at 2000 rpm for 24
minutes at room temperature with low acceleration and without
break. Afterwards the PBMCs were collected from the interface,
washed three times with PBS, resuspended in 10 mL of PBS and cells
were analyzed for cell type and number with a Beckman Coulter cell
counter AcT.TM. 5diff OV (Beckman Coulter, Cat. No. 6605580). Prior
to the B cell isolation from the PBMCs, the CD14-positive fraction
was removed by magnetic labeling of the CD14-positive cells with
CD14 microbeads (Miltenyi, Cat. No. 130-050-201) and subsequent
isolation with the autoMACS.RTM. Pro Separator (Miltenyi, Cat. No.
130-092-545). The CD14-negative fraction was used for subsequent B
cell isolation with the Miltenyi B cell isolation kit II (Cat. No.
130-091-151) and autoMACS.RTM. separation. 1.times.10.sup.5B cells
were added in 50 .mu.l of R10 medium per well to the NIH/3T3 cells.
FAP-targeted anti-human CD40 antibodies were added in 50 .mu.l of
R10 medium to the B cells at concentrations ranging from 1 .mu.g/mL
to 0.3 ng/mL (3.times. dilution series). As positive control, the
FAP-independent agonistic anti-human CD40 antibodies RO7009789
(IgG2, INN: Selicrelumab) and SGN-40 (IgG1, INN: Dacetuzumab) were
used. Both antibodies are bivalent for CD40. Since it is described
in the literature that the SGN-40 antibody requires Fc receptor
cross-linking for biological activity (C. Law et al., Cancer Res
2005, 65, 8331-8338), a mechanism that is as well under discussion
for RO7009789 (R. Dahan et al., Cancer Cell 2016, 29, 1-12), these
two antibodies were incubated with a cross-linking goat anti-human
IgG Fc.gamma. fragment specific F(ab').sub.2 fragment (Jackson
ImmunoResearch, Cat. No. 109-006-008) for 30 minutes before
addition to the B cells. After 48 hours cells were transferred into
a 96-well round-bottom plate, washed once with PBS and incubated
with 50 .mu.l of 3 .mu.g/mL of Fc receptor blocking Mouse IgG
Isotype Control (ThermoFisher Scientific, Cat. No.10400C) in PBS.
After 15 minutes of incubation at 4.degree. C., cells were washed
with PBS and 50 .mu.l of a mixture of fluorescently labelled
antibodies in PBS was added to the cells. The following
fluorescently labelled antibodies were used: anti-human CD83 BV421
(Biolegend, clone HB15e, Cat. No. 305324), anti-human CD80 BV605
(BD Biosciences, clone L307.4, Cat. No. 563315), anti-HLA-ABC FITC
(BD Biosciences, clone G46-2.6, Cat. No. 555552), anti-human CD14
PerCP-Cy5.5 (Biolegend, clone HCD14, Cat. No. 325622), anti-human
CD3 PerCP-Cy5.5 (Biolegend, clone UCHT1, Cat. No. 300430),
anti-human CD70 PE (Biolegend, clone 113-16, Cat. No. 355104),
anti-human CD86 PE-CF594 (BD Biosciences, clone FUN-1, Cat. No.
562390), anti-HLA-DR APC (BD Biosciences, clone G46-6, Cat. No.
559866) and anti-human CD19 APC-H7 (BD Biosciences, clone SJ25C1,
Cat. No. 560177). In order to distinguish between live and dead
cells, the viability dye Zombie Aqua.TM. (Biolegend, Cat. No.
423102) was added to the antibody mixture. After 30 minutes of
incubation at 4.degree. C., cells were washed twice with PBS and
then resuspended in 200 .mu.l of PBS. Cells were analyzed the same
day using a 5-laser LSR-Fortessa (BD Bioscience with DIVA
software). Data analysis was performed using the FlowJo version 10
software (FlowJo LLC). Live (aqua negative) cells, negative for
CD14 and CD3 and positive for CD19 were analyzed for CD70, CD80,
CD83 and CD86 expression.
[0748] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG.
3G, and FIG. 3H show the FAP-dependent upregulation of B cell
activation markers CD70 (FIG. 3A and FIG. 3B), CD80 (FIG. 3C and
FIG. 3D), CD83 (FIG. 3E and FIG. 3F) and CD86 (FIG. 3G and FIG. 3H)
by bispecific antigen binding molecules tetravalent for human CD40
and either mono- or bivalent for FAP. The bispecific antibody
monovalent for FAP induced a similar increase of activation marker
expression as the molecule with two FAP binding moieties. With
NIH/3T3-FAP cells upregulation of the B cell activation markers by
the bispecific antigen binding molecules was comparable to the
upregulation induced by the FAP-independent positive control
antibodies. Without FAP present (NIH/3T3-wt cells) no increase of B
cell activation markers was observed with the bispecific antigen
binding molecules, while positive control antibodies induced an
upregulation in the expression of these markers.
2.1.2 Activation of Human Daudi Cells by FAP-Targeted Anti-Human
CD40 Binding Molecules using FAP-Coated Dynabeads.RTM. as Source of
Antigen
[0749] 1.times.10.sup.5 Daudi cells in 100 .mu.l of 1.times. DMEM
plus 10% FBS were added per well of a 96-well flat-bottom plate.
Instead of using cells expressing FAP as source of antigen,
streptavidin Dynabeads.RTM. (ThermoFisher Scientific, Cat. No.:
11205D) were coated with biotinylated mouse FAP (produced in-house)
(binding capacity of 6.5.times.10.sup.4 beads: 0.01 .mu.g of
protein) according to the manufacturer's instructions and added to
the Daudi cells in a bead:cell ratio of 2:1 in 50 .mu.l of R10
medium. Usage of beads coated with FAP instead of FAP-expressing
cells provides a more stable and reproducible system, since
fluctuating quality of the cells and secretion of cellular products
that might influence APC activation status represent factors that
potentially distort results. As control non-coated beads were added
to the Daudi cells. FAP-targeted anti-human CD40 antibodies or
positive control antibodies (described in section 2.1.1) were added
in 50 .mu.l of R10 medium to the Daudi cells. After 2 days Daudi
cells were analyzed by FACS following the staining and analysis
procedures specified in 2.1.1.
[0750] B cells analyzed after 2 days of incubation with agonistic
anti-CD40 antibodies showed an increase in CD70 expression for all
antibodies (see FIG. 20A and FIG. 20B). The EC.sub.50 values of
specific molecules are summarized in Table 9 below. Upregulation of
these expression markers was dependent on FAP in case of the
different FAP-targeted antibodies and increase of expression
induced by these FAP-dependent antibodies was higher compared to
the increase induced by the cross-linked CD40 antibody (P1AD4470).
In the absence of FAP (uncoated beads) no increase of CD70 was
observed with the depicted bispecific antibodies mono- or bivalent
for CD40, while tetravalent CD40 binding molecules induced an
upregulation of CD70, but to a lesser extent than in the presence
of FAP indicating a low but detectable FAP-independent CD40
activation of tetravalent CD40 binder in Daudi cells.
TABLE-US-00012 TABLE 9 Activation of human Daudi cells using
FAP-coated Dynabeads .RTM. Molecule EC.sub.50 [nM] P1AD4470 CD40
IgG1 0.756 P1AE0637 CD40 .times. FAP 4 + 1 with C- 0.029 terminal
crossfab P1AD4453 CD40 .times. FAP 4 + 1 0.015 P1AD4574 CD40
.times. DP47 4 + 1 0.166 P1AD4455 CD40 .times. FAP 4 + 2 0.039
P1AD4465 CD40 .times. DP47 4 + 2 n/a P1AA9641 CD40 .times. FAP 2 +
1 0.068 P1AE0408 CD40 .times. FAP 2 + 1 head-to-tail 0.094 P1AA9663
CD40 .times. FAP 2 + 2 0.124 P1AE0192 CD40 .times. FAP 1 + 1 0.409
P1AE0889 CD40 (VH2a/VF2a) .times. FAP 0.055 4 + 1 with C-terminal
crossfab P1AE0890 CD40 (VH2a/VF2a) .times. FAP 0.058 4 + 1 with
C-terminal crossfab
2.1.3 Activation of Human B Cells by FAP-Targeted Anti-Human CD40
Binding Molecules using FAP-Coated Dynabeads.RTM. as Dource of
Antigen
[0751] B cells were isolated from buffy coats as described in
section 2.1.1 and 1.times.10.sup.5B cells in 100 .mu.l of R10
medium were added per well of a 96-well flat-bottom plate.
Streptavidin Dynabeads.RTM. (ThermoFisher Scientific, Cat.
No.:11205D) were coated with biotinylated human or mouse FAP
(produced in-house) (binding capacity of 6.5.times.10.sup.4 beads:
0.01 .mu.g of protein) according to the manufacturer's instructions
and added to the B cells in a bead:cell ratio of 2:1 in 50 .mu.l of
R10 medium. As control non-coated beads were added to the B cells.
The FAP-targeted anti-human CD40 antibodies or positive control
antibodies (described in section 2.1.1) were added in 50 .mu.l of
R10 medium to the B cells. After 2 days B cells were analyzed by
FACS following the staining and analysis procedures specified in
2.1.1. Alternatively B cells were cultured for five days in
presence of the agonistic anti-CD40 antibodies and 110 .mu.l
supernatant were taken for IL-6 measurement using the human IL-6
DuoSet ELISA kit (R&D, Cat. No. DY206-05). It is known that B
cells produce increased amounts of IL-6 upon stimulation with the
CD40 ligand without the need of additional B cell receptor stimuli
(M. Duddy et al., J. Immunol. 2004, 172, 3422-3427). The ELISA was
performed as described in the protocol provided by the manufacturer
with the only difference of using half of the recommended amounts
for every step of the assay. B cells were analyzed by FACS
following the staining and analysis procedures specified in section
2.1.1.
[0752] B cells analyzed after 2 days of incubation with agonistic
anti-CD40 antibodies showed an increase in CD70, CD83 and CD86
expression for all antibodies (see FIG. 4A, FIG. 4B, FIG. 4C, FIG.
4D, FIG. 4E, FIG. 4F, FIG. 21A and FIG. 21B and FIG. 25A, FIG. 25B,
FIG. 25C, FIG. 25D). The EC.sub.50 values relating to the increase
of CD86 expression of specific molecules are summarized in Table 10
below. Upregulation of these expression markers was dependent on
FAP in case of the different FAP-targeted antibodies and increase
of expression induced by these FAP-dependent antibodies was
comparable or slightly lower to the increase induced by the
cross-linked CD40 antibody P1AD4470.
TABLE-US-00013 TABLE 10 Activation of human B cells using
FAP-coated Dynabeads .RTM. shown as increase of CD86 expression
Molecule EC.sub.50 [nM] P1AD4470 CD40 IgG1 0.072 P1AE0637 CD40
.times. FAP 4 + 1 with C- 0.202 terminal crossfab P1AD4453 CD40
.times. FAP 4 + 1 0.280 P1AD4574 CD40 .times. DP47 4 + 1 n/a
P1AD4455 CD40 .times. FAP 4 + 2 0.325 P1AD4465 CD40 .times. DP47 4
+ 2 n/a P1AA9641 CD40 .times. FAP 2 + 1 0.909 P1AE0408 CD40 .times.
FAP 2 + 1 head-to-tail 0.658 P1AA9663 CD40 .times. FAP 2 + 2 1.004
P1AE0192 CD40 .times. FAP 1 + 1 0.742 P1AE0889 CD40 (VH2a/VF2a)
.times. FAP 0.329 4 + 1 with C-terminal crossfab P1AE0890 CD40
(VH2a/VF2a) .times. FAP 0.345 4 + 1 with C-terminal crossfab
[0753] After 5 days of B cell incubation with FAP-coated
Dynabeads.RTM., antigen binding molecules targeting human CD40 and
FAP induced a FAP-dependent upregulation of CD80 expression on B
cells. The levels of CD80 expression induced by anti-human CD40
antibodies with either one or two FAP binding sites were
comparable. Treatment of B cells with positive control anti-CD40
antibodies led to a similar extent of CD80 upregulation. Elevated,
FAP-dependent CD86 expression could be as well detected with the
bispecific antigen binding molecules. Again, presence of one or two
FAP binding sites made no major difference in expression levels of
this activation marker. Compared to the FAP-independent
upregulation of CD86 induced by cross-linked SGN-40 (dacetuzumab)
or agonistic CD40 antibody selicrelumab (RO 7009789), CD86
upregulation induced by FAP-dependent bispecific antigen binding
molecules was slightly lower. For CD70 and CD83 no or only very
limited upregulation was observed with the bispecifc antibodies
targeting FAP and CD40, while the positive control antibodies
clearly showed an effect on these B cell activation markers (FIG.
5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG.
5H).
[0754] FIG. 6A and FIG. 6B show the effects of different agonistic
anti-CD40 antibodies on B cell IL-6 production. With FAP present
IL-6 concentration in the supernatants were elevated to a similar
extent for all agonistic anti-human CD40 antibodies tested (about
6-fold increase compared to untreated (UT) conditions). Upon
incubation of the B cells with non-coated Dynabeads.RTM. no
increase in IL-6 production was detected with the bispecific
antigen binding molecules, demonstrating FAP-dependency of these
molecules.
2.1.4 Activation of Human Monocyte-Derived DCs (moDCs) by
FAP-Targeted Aanti-Human CD40 Binding Molecules using Human
FAP-Coated Dynabeads.RTM. as Source of Antigen
[0755] PBMCs were isolated from buffy coats by Lymphoprep.TM.
density centrifugation. Subsequently monocytes were isolated with
CD14 microbeads and autoMACS.RTM. separation as described in 2.1.1.
In order to generate monocyte-derived DCs (moDCs), 3.times.10.sup.6
monocytes were seeded per well of a 6 well plate (TPP, Cat. No.
92006) with a density of 1.times.10.sup.6 cells per mL. For DC
maturation moDC medium consisting of RPMI 1640 supplemented with 1%
(v/v) Penicillin Streptomycin, 2% (v/v) human serum (heat
inactivated for 30 minutes at 56.degree. C.) (Sigma-Aldrich, Cat.
No. H4522, Lot. No. SLBP1687V), 20 ng/mL freshly added recombinant
human granulocyte macrophage colony-stimulating factor (GM-CSF)
(Peprotech, Cat. No. 300-03-20UG, Lot No. 081230H1213) and 20 ng/mL
of freshly added recombinant human IL-4 (Peprotech, Cat. No.
200-04-100UG, Lot. No. 09151403116) was used. After five days moDCs
were harvested from the 6-well plates by gentle removal of
suspension and semi-adherent cells and resuspended in fresh moDC
medium containing human IL-4 and GM-CSF. 2.times.10.sup.5 moDCs
were seeded in 100 .mu.l of moDC medium per well of a 96-well
flat-bottom plate. Streptavidin Dynabeads.RTM. were coated with
biotinylated human FAP and added in 50 .mu.l in a bead/DC ratio of
2:1 (as described in 2.1.3). As control non-coated beads were added
to the moDCs. FAP-targeted anti-human CD40 antibodies were added in
50 .mu.l to the DCs at concentrations ranging from 1 .mu.g/mL to
0.3 ng/mL (3.times. dilution series). As positive, FAP-independent
controls the agonistic anti-human CD40 antibody RO7009789 and the
cross-linked CD40 antibody were used. Two days after addition of
the Dynabeads.RTM. and of the different agonistic anti-human CD40
antibodies moDC activation was measured by FACS. FACS staining was
performed as specified in 2.1.1 using a mixture of fluorescently
labelled antibodies consisting of anti-human CD86 BV421 (BD
Biosciences, clone FUN-1, Cat. No. 562432), anti-human CD80 BV605
(BD Biosciences, clone L307.4, Cat. No. 563315), anti-HLA-ABC FITC
(BD Biosciences, clone G46-2.6, Cat. No. 555552), anti-human CD1c
PerCp-Cy5.5 (BD Biosciences, clone F10/21A3, Cat. No. 565424),
anti-human CD70 PE (Biolegend, clone 113-16, Cat. No. 355104),
anti-human CD11c PE-eF610 (eBioscience, clone 3.9, Cat. No.
61-0116-42), anti-human CD83 PE-Cy7 (BD Biosciences, clone HB15e,
Cat. No. 561132), anti-human CD209 APC (BD Biosciences, clone
DCN46, Cat. No. 551545), anti-human CD3 Alexa Fluor 700
(eBioscience, clone OKT3, Cat. No. 56-0037-42), anti-human CD14
APC-H7 (BD Biosciences, clone M5E2, Cat. No. 561384) and viability
dye Zombie Aqua.TM. (Biolegend, Cat. No. 423102). Cells were
analyzed the same day using a 5-laser LSR-Fortessa. Data analysis
was performed using the FlowJo version 10 software. Single, live
cells were gated for CD3 negative and CD14 negative cells. Based on
this population CD lc and CD11c positive cells were analyzed for
the expression of the activation markers CD70, CD80, CD83 and
CD86.
[0756] For all agonistic anti-human CD40 antibodies a pronounced
and similar upregulation of CD83 on moDCs could be observed (FIG.
7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F). In case of the
FAP-dependent anti-CD40 antibodies, this upregulation was detected
in a FAP-dependent manner. With the bispecifc antigen binding
molecules targeting CD40 and FAP, CD80 expression was only slightly
increased, however this increase was FAP-dependent and comparable
to the CD80 upregulation induced by positive control antibody
RO7009789 on the DCs (FIG. 7C and FIG. 7D). While CD86 expression
was not significantly changed on DCs incubated with the different
anti-CD40 antibodies compared to untreated DCs (FIG. 7G and FIG.
7H), CD70 expression was elevated due to the agonistic effects of
these antibodies (FIG. 7A and FIG. 7B). Again both FAP-dependent
antibodies only showed activating properties when FAP was present
and their effects on CD70 were similar. However numbers of moDCs
with upregulated CD70 expression was in a low range (maximum 8% of
CD11c and CD1c positive cells). RO7009789 showed a higher potency
to upregulate CD70 on DCs compared to the bispecific molecules in
this experimental setting.
2.1.5 Activation of HEK-Blue.TM. CD40L Cells by FAP-Targeted
Anti-Human CD40 Binding Molecules using Murine FAP-Coated
Dynabeads.RTM. as Source of Antigen
[0757] HEK-Blue.TM. CD40L cells (InvivoGen, Cat.No. hkb-cd40) were
used as a reporter cell line to analyze human CD40 stimulation
mediated by FAP-targeted anti-human CD40 binding molecules.
HEK-Blue.TM. CD40L cells stably express the human CD40 receptor and
NF.kappa.B-inducible secretion of embryonic alkaline phosphatase
(SEAP). Binding of CD40L or agonistic anti-CD40 antibody to the
CD40 receptor expressed on HEK-Blue.TM. CD40L cells triggers a
signaling cascade leading to NFKB-mediated SEAP production. The
amount of produced SEAP directly correlates with the extent of CD40
receptor activation. The levels of secreted SEAP in the supernatant
can be measured with a spectrophotometer at 620-655 nm.
0.5.times.10.sup.5 HEK-Blue.TM. CD40L cells in 160 .mu.l pre-warmed
HEK-Blue.TM. detection medium (InvivoGen, Cat. No. hb-det2) were
seeded per well of a 96-well flat-bottom plate (TPP, Cat. No.
92696). Streptavidin Dynabeads.RTM. were coated with biotinylated
murine FAP and added in 20 .mu.l PBS in a bead:cell ratio of 2:1
(as described in 2.1.3). As control non-coated beads were added to
the reporter cells. FAP-targeted anti-human CD40 antibodies were
added in 20 .mu.l PBS to the cells at concentrations ranging from
6.89 nM to 0.0032 nM (3.times. dilution series). As control
molecules antibodies tetravalent for human CD40 with one or two
DP47 domains instead of a FAP binding domain and a FAP-independent
cross-linked CD40 antibody (P1AD4470) were used. After 8 hours
incubation at 37.degree. C. SEAP levels in the supernatant were
measured by a spectrophotometer at 650 nm.
[0758] For all agonistic FAP-targeted anti-human CD40 antibodies a
pronounced and comparable SEAP production was observed. In the case
of FAP-targeted bispecific antibodies mono- or bivalent for human
CD40 SEAP production was detected only in the presence of FAP. In
contrast, reporter cells treated with FAP-targeted bispecific
antibodies tetravalent for human CD40 secreted SEAP independently
of FAP availability. However, in the presence of FAP higher SEAP
levels were detected in supernatant of reporter cells treated with
these antibodies. Moreover, the negative control antibodies
tetravalent for human CD40 with one or two DP47 domains instead of
a FAP binding domain induced comparable SEAP production in
HEK-Blue.TM. CD40L cells in the presence and absence of FAP. The
positive control antibody CD40 IgG (P1AD4470) +F(ab) induced
similar levels of SEAP production as compared to FAP-targeted
bispecific antibodies bivalent or tetravalent for human CD40 in the
presence of FAP-coated beads (FIG. 8A, FIG. 8B, FIG. 19A and FIG.
19B). The EC.sub.50 values as measured for the CD40 antibody as
well as for different bispecific antibodies are shown in Table 11
below.
TABLE-US-00014 TABLE 11 Activation of HEK-Blue .TM. CD40L cells
using murine FAP-coated Dynabeads .RTM. Molecule EC.sub.50 [nM]
P1AD4470 CD40 IgG1 PGLALA 0.125 P1AE0637 CD40 .times. FAP 4 + 1
with C- 0.031 terminal crossfab P1AD4453 CD40 .times. FAP 4 + 1
0.067 P1AD4574 CD40 .times. DP47 4 + 1 0.421 P1AD4455 CD40 .times.
FAP 4 + 2 0.077 P1AA9641 CD40 .times. FAP 2 + 1 0.197 P1AE0408 CD40
.times. FAP 2 + 1 head-to-tail 0.167 P1AA9663 CD40 .times. FAP 2 +
2 0.238 P1AE0192 CD40 .times. FAP 1 + 1 0.639 P1AE0889 CD40
(VH2a/VF2a) .times. FAP 0.044 4 + 1 with C-terminal crossfab
P1AE0890 CD40 (VH2a/VF2a) .times. FAP 0.0005 4 + 1 with C-terminal
crossfab
2.2 CD40-Mediated Activation of DCs by FAP-Targeted Anti-CD40
Binding Molecules and Subsequent Priming of T Cells
[0759] In order to demonstrate the ability of DCs activated by the
FAP-dependent anti-human CD40 antibodies to efficiently prime T
cells, in vitro T cell priming assays were established. For these
assays DCs from the spleens of transgenic mice expressing the human
CD40 receptor (huCD40tg mice) (mice with similar human and murine
CD40 receptor expression pattern; C57BL/6 background; generated by
Taconic) were isolated, pulsed with either SIINFEKL peptide or with
ovalbumin (OVA) (DEC-205 receptor-mediated antigen uptake) and
incubated with different agonistic anti-human CD40 antibodies. FAP
was provided via FAP-coated Dynabeads.RTM. in order to show
FAP-dependency of the bispecific antigen binding molecules. 24
hours later CD8 positive T cells were isolated from spleens of OT1
mice (CD8-positive T cells of these mice all possess a transgenic
TCR recognizing SIINFEKL in the context of H2-Kb;
C57BL/6-Tg(TcraTcrb)1100Mjb/Crl, Charles River), carboxyfluorescein
succinimidyl ester (CFSE) labelled and added to the pulsed DCs. On
day five of the experiment s DC and T cell cytokines were analyzed
in the supernatants of the cultured cells and T cells were analyzed
for activation and proliferative capabilities.
2.2.1 T Cell Priming via SIINFEKL-Pulsed DCs Activated by
FAP-Targeted Anti-CD40 Binding Molecules
[0760] DCs were isolated from the spleens of huCD40tg mice. In
order to isolate splenic DCs, the spleen from a huCD40tg mouse was
put into one well of a 6-well plate containing 2.25 mL Hank's
Balanced Salt Solution (HBSS) with Calcium.sup.2+(gibco, Cat. No.
14025-05), 250 .mu.l of a 10 mg/mL solution of collagenase D (end
concentration 1 mg/mL) (Sigma-Aldrich, Cat. No. 11088866001) and
12.5 .mu.l of a 10 mg/mL DNase solution (end concentration 0.05
mg/mL) (Sigma-Aldrich, D5025-150KU, Lot. No. SLBRO535V). Using a 3
mL syringe (BD, Cat. No. 309658) with a 21G needle (Braun, Cat. No.
4657527) the spleen was ballooned and subsequently, with the help
of scissors, torn into small pieces. After a 25 minutes incubation
at 37.degree. C., 50 .mu.L of 0.5 M ethylenediaminetetraacetic acid
(EDTA) (Applichem, Cat. No. A4892.1000) were added, followed by a
second incubation step at 37.degree. C. for five minutes. The
solution containing splenocytes and small pieces of splenic tissue
was filtered through a 40 .mu.m filter (Corning, Cat. No. 352340)
into a 50 mL polypropylene centrifuge tube. Splenic tissue pieces
were smashed through the filter with the end of a 3 mL syringe
plug. In the next step the 50 mL tube was centrifuged at 1500 rpm
for 5 minutes at room temperature, the supernatant was discarded
and 1 mL of 1.times. cell lysis buffer (diluted 1:10 with distilled
water) (BD, Cat. No. 555899) was added to the splenocytes in order
to lyse the red blood cells. After four minutes of incubation at
room temperature, 20 mL of R10 were added followed by a
centrifugation step at 1500 rpm for 5 minutes at room temperature.
The supernatant was removed, the splenocytes were resuspended in 30
mL of R10 and cell count as well as viability were determined with
the automated EVE cell counter (VWR, Cat. No. 734-2675). The mouse
CD11 c UltraPure microbeads (Miltenyi, Cat. No. 130-108-338) were
used according to the manufacturer's instruction to isolate DCs by
autoMACS.RTM. separation. Subsequently 0.25.times.10.sup.5 DCs were
seeded in 50 .mu.l of R10 per well of a 96-well flat-bottom plate.
The DCs were then pulsed with SIINFEKL peptide (Ovalbumin residues
257-264) (Eurogentec, Cat. No. AS-60193-5, Lot. No. 1360618) in a
suboptimal concentration of 1 pg/mL. This limited amount of antigen
allows detecting variances in T cell activation due to differently
activated DCs. As a positive control in order to induce high T cell
activation independent of additional DC activating stimuli,
SIINFEKL was added at a concentration of 1 ng/mL to the DCs. DCs
that were not pulsed with the SIINFEKL antigen served as negative
control. Human FAP-coated or non-coated Dynabeads.RTM. were added
in 50 .mu.L of R10 to the DCs at a 2:1 bead:cell ratio. In the next
step different agonistic anti-CD40 antibodies were added in 50
.mu.L of R10 at concentrations ranging from 1 .mu.g/mL to 1 ng/mL
(10.times. dilution series). In this experimental setup, the
bispecific anti-human CD40 antibody containing two FAP binding
sites was compared to its equivalent with two DP47 domains instead
of FAP binding domains, to the FAP-independent RO7009789 and
cross-linked SGN-40 and to a murine FAP-dependent bispecific
antibody tetravalent for murine CD40 and bivalent for FAP (28H1 FAP
binder). After 24 hours splenic CD8-positive cells from OT1 mice
were isolated. In order to do so, the spleen of an OT1 mouse was
smashed through a 40 .mu.m filter with the end of a 3 mL syringe
plug into a 50 mL tube. The filter was washed with R10 and the
splenocytes were centrifuged at 1500 rpm for 5 minutes at room
temperature. 1 mL of 1.times. cell lysis buffer (diluted 1:10 with
distilled water) was added to the cells and after four minutes of
incubation at room temperature, 20 mL of R10 were added. The tube
was centrifuged at 1500 rpm for 5 minutes at room temperature and
the supernatant was discarded. The splenocytes were resuspended in
30 mL of R10 and cell counts as well as viability were determined
with the automated EVE cell counter. CD8-positive cells were
isolated in a negative selection process using the mouse CD8a.+-.T
Cell Isolation Kit (Miltenyi, Cat. No. 130-104-075) and
autoMACS.RTM. separation according to the manufacturer's
instructions. CD8-positive cells that were found in the negative
fraction after the separation were then washed with pre-warmed PBS,
counted with the EVE cell counter and the cell number was adjusted
to 2.times.10.sup.7 cells/mL in pre-warmed PBS. 10 mM CFSE solution
(CellTrace.TM. CFSE Cell Proliferation Kit, ThermoFisher, Cat. No.
C34554) was 5000-fold diluted in pre-warmed PBS and added to the
cells resuspended in PBS in a 1:1 ratio (CFSE end concentration 1
.mu.M). After a short vortex, cells were incubated for five minutes
at room temperature. The labelling reaction was stopped by adding
40 mL of pre-warmed R10 medium to the cells. After two washing
steps with PBS, CD8-positive cells were resuspended in R10 and
0.5.times.10.sup.5 cells were added in 100 .mu.l R10 to the pulsed
DCs. On day five of the experiment T cells were restimulated for
intracellular cytokine staining (ICS) with 0.5 .mu.g/mL of SIINFEKL
and 2 .mu.g/mL anti-mouse CD28 antibody (eBioscience, clone 37.51,
Cat. No. 16-0281-86). One hour after SIINFEKL and anti-CD28
addition, Brefeldin A (BFA) (BD, Cat. No. 51-2301KZ) (1:1000) was
added to the cells in order to block intracellular protein
transport. After another four hours incubation step, 150 .mu.l of
the supernatant were taken for Luminex.TM. based multiplexed
cytokine measurement. In order to measure a set of 23 cytokines,
the Bio-Plex Pro.TM. Mouse Cytokine GrpI Panel 23-Plex kit (BioRad,
Cat. No. M60009RDPD) was used according to the manufacturer's
instructions. For flow cytometry analysis of the T cells, cells in
the 96-well flat-bottom plates were transferred into 96-well
round-bottom plates, washed once with PBS and incubated with 50
.mu.l of 3 .mu.g/mL of Fc receptor blocking Mouse IgG Isotype
Control in PBS. After 15 minutes of incubation at 4.degree. C.,
cells were washed with PBS and 50 .mu.l of a mixture of
fluorescently labelled antibodies in PBS was added to the cells.
The following antibodies were used: anti-mouse CD86 BV785
(Biolegend, clone GL-1, Cat. No. 105043), anti-I-A/I-E PerCp-Cy5.5
(Biolegend, clone M5/114.15.2, Cat. No. 107626), anti-mouse CD70 PE
(eBioscience, clone FR70, Cat. No. 12-0701-82), anti-mouse CD3
PE-CF594 (BD Biosciences, clone 145-2C11, Cat. No. 562286),
anti-mouse CD25 PE-Cy7 (eBioscience, clone PC61.5, Cat. No.
25-0251-82), anti-mouse CD11c APC (BD Biosciences, clone HL3, Cat.
No. 561119), anti-mouse CD44 Alexa Fluor 700 (BD Biosciences, clone
IM7, Cat. No. 560567) and anti-mouse CD8 APC-Cy7 (Biolegend, clone
53-6.7, Cat. No. 100714). In order to distinguish between live and
dead cells, the viability dye Zombie Aqua.TM. was added to the
antibody mixture. Cells were incubated for 30 minutes at 4.degree.
C. with the extracellular staining antibody solution. Afterwards
cells were washed two times with PBS, permeabilized and
intracellularly stained for IFN.gamma. using anti-mouse IFN.gamma.
BV421 (Biolegend, clone XMG1.2, Cat. No. 505830) with the
Foxp3/Transcription Factor Staining Buffer Set (eBioscience, Cat.
No. 00-5523-00) according to the manufacturer's protocol. Cells
were resuspended in 200 .mu.l of PBS and analyzed the same day
using a 5-laser LSR-Fortessa. Data analysis was performed using the
FlowJo version 10 software. The population of live cells that
displayed expression of CD8 and CD3 was analyzed for CFSE dye
dilution, IFN.gamma. production, CD44 and CD25 expression.
[0761] FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG.
9G, and FIG. 9H show that DCs pulsed with low amounts of SIINFEKL
and stimulated with different agonistic anti-CD40 antibodies are
able to induce T cell proliferation. In case of the FAP-dependent
bispecific anti-CD40 antibodies proliferation is only induced when
FAP is provided in the assay. Levels of proliferation induced by
DCs stimulated with the murine or the human version of the
bispecific antigen binding molecules with four CD40 and two FAP
binding moieties was comparable. This strongly suggests that
downstream signaling of the human CD40 receptor expressed on DCs is
not impaired in the huCD40tg mice. No significant upregulation of
the T cell activation markers CD44 and CD25 or IFN.gamma.
production was observed for T cells cocultured with DCs that have
been stimulated with different agonistic anti-CD40 antibodies. Only
DCs pulsed with high amounts of SIINFEKL displayed clear changes of
these markers compared to the untreated condition.
[0762] Cytokine concentration measurement in the supernatant showed
an effect of the agonistic anti-CD40 antibodies on IL-2 (FIG. 9I)
and IL-12(p40) (FIG. 9J) expression. With the FAP-dependent human
anti-CD40 antibody elevated IL-12(p40) levels were detected only in
the presence of FAP. However, the murine equivalent bispecific
antigen binding molecule induced a markedly higher secretion of
IL-12(p40). IL-2 secretion was increased to a similar extent with
both, the anti-human CD40 and the anti-mouse CD40 bispecific
antigen binding molecules in a FAP-dependent way.
2.2.2 T Cell Priming Via OVA-Pulsed DCs Activated by FAP-Targeted
Anti-CD40 Binding Molecules
[0763] DCs were isolated from the spleens of huCD40tg mice and
FAP-coated or non-coated Dynabeads.RTM. as well as different
agonistic anti-CD40 antibodies were added to the splenic DCs as
described in section 2.2.1. Instead of pulsing DCs with SIINFEKL,
which requires no uptake and processing by the DCs, OVA protein was
used as antigen. In order to promote the OVA uptake in a Toll-like
receptor (TLR) stimulus independent way (additional TLR stimuli
might lead to a high overall activation of DCs, making the
detection of different activation states due to stimulation with
agonistic anti-CD40 antibodies impossible) the Ova Antigen Delivery
Reagent (Miltenyi, Cat. No. 130-094-663) in combination with a
biotinylated anti-mouse DEC205 antibody (Miltenyi, clone NLDC-145,
Cat. No. 130-101-854) was used according to the manufacturer's
protocol. In brief, DCs are incubated with a biotinylated antibody
that binds to the DEC205 receptor, which is highly expressed on
CD8-positive cross-presenting DCs (M. Lahoud et al., Int Immunol.
2000, 12(5), 731-735). Afterwards the Ova delivery reagent, an
anti-biotin antibody coupled to FITC and OVA, is added to the cells
leading to DEC205 receptor-mediated uptake of OVA. In order to
provide a negative control, DCs were only labelled with the
anti-DEC205 antibody without the addition of OVA. On the next day,
CD8-positive T cells were isolated from OT1 mice, CFSE labelled and
added to the DCs as described in section 2.2.1. On day five of the
experiment 150 .mu.l of supernatant were taken for IFN.gamma.
measurement using the Mouse IFN-gamma DuoSet ELISA kit (R&D,
Cat. No. DY485-05). The ELISA was performed as described in the
protocol provided by the manufacturer. For FACS analysis of the T
cells, cells in the 96-well flat-bottom plates were transferred
into 96-well round-bottom plates, washed once with PBS and
incubated with 50 .mu.l of 3 .mu.g/mL of Fc receptor blocking Mouse
IgG Isotype Control in PBS. After 15 minutes of incubation at
4.degree. C. cells were washed with PBS and 50 .mu.l of a mixture
of fluorescently labelled antibodies in PBS were added to the
cells. The following antibodies were used: anti-mouse CD4 BV421
(Biolegend, clone GK1.5, Cat. No. 100438), anti-mouse CD86 BV785
(Biolegend, clone GL-1, Cat. No. 105043), anti-I-A/I-E
PerCp-Cy5.5
[0764] (Biolegend, clone M5/114.15.2, Cat. No. 107626), anti-mouse
CD70 PE (eBioscience, clone FR70, Cat. No. 12-0701-82), anti-mouse
CD3 PE-CF594 (BD Biosciences, clone 145-2C11, Cat. No. 562286),
anti-mouse CD25 PE-Cy7 (eBioscience, clone PC61.5, Cat. No.
25-0251-82), anti-mouse CD11c APC (BD Biosciences, clone HL3, Cat.
No. 561119), anti-mouse CD44 Alexa Fluor 700 (BD Biosciences, clone
IM7, Cat. No. 560567) and anti-mouse CD8 APC-Cy7 (Biolegend, clone
53-6.7, Cat. No. 100714). In order to distinguish between live and
dead cells, the viability dye Zombie Aqua.TM. was added to the
antibody mixture. Cells were incubated for 30 minutes at 4.degree.
C. with 50 .mu.l of the staining antibody mix. Afterwards cells
were washed two times with PBS, resuspended in 200 .mu.l of PBS and
analyzed using 5-laser LSR-Fortessa. Data analysis was performed
using the FlowJo version 10 software. Viable CD3- and CD8-positive
cells were analyzed for CFSE signal, CD25 and CD44 expression.
[0765] FIG. 10A, FIG. 10B, FIG. 10C, FIG. 11A, FIG. 11B, FIG. 11C,
FIG. 12A, FIG. 12B, FIG. 12C and FIG. 22A, and FIG. 22B show that
DCs incubated with the OVA delivery reagent and stimulated with the
bispecific antigen binding molecule targeting human CD40 and FAP
can significantly enhance CD8 positive OT1 T cell proliferation
(FIG. 10A, FIG. 10B, FIG. 10C and FIG. 22A, FIG. 22B) as well as
expression of the T cell activation markers CD25 (FIG. 11A, FIG.
11B, and FIG. 11C) and CD44 (FIG. 12A, FIG. 12B, and FIG. 12C).
These effects were FAP-dependent. The results of the IFN.gamma.
ELISA confirm the enhanced T cell activation due to the human
anti-CD40 FAP-targeted antibody: IFN.gamma. levels were elevated in
conditions with T cells cocultured with DCs treated with the
anti-human CD40 FAP targeting antibody (FIG. 13A, FIG. 13B, FIG.
13C). Effects of the murine anti-CD40 FAP-targeted antibody were
comparable, underpinning the hypothesis that the huCD40tg mouse
model provides a suitable system for measuring the effects of
agonistic anti-human CD40 antibodies.
Example 3
Functional Properties of FAP-Targeted Anti-Murine CD40 Binding
Molecules
3.1 CD40-Mediated In Vitro Activation of Murine B Cells by
FAP-Targeted Anti-Murine CD40 Binding Molecules
[0766] Spleens from C57BL/6J mice were processed as described in
section 2.2.1. Murine B cells were isolated from splenocytes using
the mouse B cell isolation kit (Miltenyi, Cat. No. 130-090-862)
according to the manufacturer's instructions. 1.times.10.sup.5 B
cells were seeded in 100 .mu.l R10 per well of a 96-well
flat-bottom plate. Dynabeads.RTM. coated with murine biotinylated
FAP (in-house production) (see 2.1.3 for detailed description) or
non-coated Dynabeads as control were added in 50 .mu.l R10 in a
bead:cell ratio of 2:1. Agonistic anti-murine CD40 antibodies were
added in 50 .mu.l of R10 to the B cells. Antibody concentrations
varied from 1 .mu.g/mL to 0.3 ng/mL (3.times. dilution series). In
this experimental setup bispecific antigen binding molecules
carrying four anti-mouse CD40 binding sites and either one or two
FAP binding sites (28H1 FAP binder, equivalent to the FAP binding
domain in anti-human CD40 bispecific antigen binding molecules)
were compared to the FAP-independent FGK4.5 antibody (rat IgG2a,
BioXcell Catalogue No. BE0016-2), which is bivalent for murine
CD40. The biological activity of FGK4.5 is dependent on Fc receptor
cross-linking (L. Richman et al., Cancer Immunol Res. 2014, 2(1)),
therefore FGK4.5 was pre-incubated for 30 minutes at room
temperature with a goat anti-rat IgG (H+L) cross-linking antibody
(Jackson ImmunoResearch, Cat. No. 112-005-003, Lot. No. 123801).
After 48 hours B cells were analyzed for expression of activation
markers by FACS. For this purpose, B cells were transferred into
96-well flat-bottom plates, washed with PBS and incubated with 50
.mu.l of 3 .mu.g/mL of Fc receptor blocking Mouse IgG Isotype
Control in PBS. After 15 minutes of incubation at 4.degree. C.,
cells were washed with PBS and 50 .mu.l of a mixture of
fluorescently labelled antibodies in PBS was added to the cells.
The following antibodies were used: anti-mouse CD19 BV605 (BD
Biosciences, clone 1D3, Cat. No. 563148), anti-mouse CD86 BV785
(Biolegend, clone GL-1, Cat. No. 105043), anti-I-A/I-E PerCp-Cy5.5
(Biolegend, clone M5/114.15.2, Cat. No. 107626), anti-mouse CD70 PE
(eBioscience, clone FR70, Cat. No. 12-0701-82), anti-mouse CD80
PE-CF594 (BD Biosciences, clone 16-10A1, Cat. No. 562504),
anti-mouse CD3 PE-Cy7 (BD Biosciences, clone 145-2C11, Cat. No.
552774), anti-mouse NK1.1 PE-Cy7 (Biolegend, clone PK136, Cat. No.
108714), anti-mouse CD11c APC (BD Biosciences, clone HL3, Cat. No.
561119), anti-mouse CD45 Alexa Fluor 700 (eBioscience, clone
30-F11, Cat. No. 56-0451-82), anti-mouse CD8 APC-Cy7 (Biolegend,
clone 53-6.7, Cat. No. 100714). To distinguish between live and
dead cells, the viability dye Zombie Aqua.TM. was added to the
antibody mixture. Cells were incubated for 30 minutes at 4.degree.
C. with the staining mixture, washed two times with PBS and then
resuspended in 200 .mu.l of PBS. FACS analysis was performed with a
5-laser LSR-Fortessa and data analysis was conducted using the
FlowJo version 10 software. Viable, single cells were gated for
CD11c-negative, CD3-negative and NK1.1-negative cells in order to
exclude non-B cells. CD8-negative, CD19-positive cells were
analyzed for expression of the B cell activation markers CD70,
CD80, CD83 and CD86.
[0767] Incubation of murine B cells with the FAP-targeted
anti-mouse CD40 antibodies with either one or two FAP binding
moieties increased expression of B cell activation markers. CD70
expression was only slightly increased with the bispecific antigen
binding molecules compared to conditions with the cross-linked
FGK4.5 antibody (FIG. 14A and FIG. 14B). However, overall numbers
of cells expressing high levels of CD70 were rather low. CD80
expression was upregulated in a FAP-dependent manner upon treatment
of B cells with the bispecific molecules targeting murine CD40 and
FAP (FIG. 14C and FIG. 14D). Bispecific molecules showed higher
potency than the bivalent FAP-independent FGK4.5 antibody in case
of CD80 upregulation. CD86 expression increase was as well induced
by all agonistic anti-CD40 antibodies tested and expression levels
were comparable for all antibodies (FIG. 14E and FIG. 14F). While
CD86 upregulation was FAP-dependent with the tetravalent anti-mouse
CD40 antibody possessing two FAP binding moieties, a
FAP-independent effect was observed for the bispecific antigen
binding molecule having only one FAP binding site. In addition, a
FAP-independent upregulation of MHC-II expression was observed for
all tested bispecific antigen binding molecules (FIG. 14G and FIG.
14H)
3.2 CD40-Mediated In Vivo Activation of Murine DCs and T Cells by
FAP-Targeted Anti-Murine CD40 Binding Molecules
[0768] The murine colon adenocarcinoma MC38_FAP transfectant tumor
cell line with mouse FAP expression was obtained from an
in-vivo-passage performed at Roche Glycart AG and after expansion
deposited in the Glycart internal cell bank. MC38_muFAP_invipa
cells were cultured in DMEM containing 10% FCS (PAA Laboratories,
Austria), 1 mM Pyruvate, 1.times. NEAA and 6 .mu.g/ml Puromycine.
Cells were cultured at 37.degree. C. in a water-saturated
atmosphere at 5% CO2 and were injected at in vitro passage 14 at a
viability of 95%. 2.times.10.sup.6 tumor cells were injected
subcutaneously in a 100 .mu.l cell suspension (50% RPMI medium and
50% matrigel). 33 C57B1/6 female mice with an age of 8-9 weeks at
start of the experiment (purchased from Charles Rivers, Germany)
were maintained under specific-pathogen-free condition with daily
cycles of 12 h light and 12 h darkness according to committed
guidelines (GV-Solas; Felasa; TierschG). Experimental study
protocol was reviewed and approved by local government (P 2011-128)
and after arrival animals were maintained for one week to get
accustomed to the new environment and for observation. They were
afterwards implanted with a transponder subcutaneously on the right
side of the back for identification and maintained one more week
for recovery. Continuous health monitoring was carried out on
regular basis. To study the FAP-targeted activation of CD40 in vivo
mice were injected subcutaneously on study day 0 with
2.times.10.sup.6 of MC38-FAP. At day x when tumors reached 200
mm.sup.3, 9 mice per group were injected i.p. with 200 .mu.L of the
different compounds. Mice in the vehicle group were injected with
Histidine buffer and animals in the treatment groups were either
injected with 10 mg/kg FGK4.5 or 15 mg/kg FGK4.5 4+1. Animals were
controlled daily for clinical symptoms and detection of adverse
effects such as weight loss. Termination criteria for animals were
clinical sickness, impaired locomotion and scruffy fur. At the time
points 72 h and 8 d post therapy injection, tumor, spleen,
tumor-draining and tumor-non-draining lymph nodes were collected
from three mice per group and analyzed by flow cytometry. In
addition, serum from all sacrificed animals was collected to
analyze serum enzymes indicative of liver injury.
[0769] For flow cytometer analysis single cell suspensions of all
collected organs were prepared and stained with fluorescently
labelled antibodies as described in section 2.1.1. and section 3.1,
respectively. To distinguish between live and dead cells, the
viability dye Zombie Aqua.TM. was added to the antibody mixture.
Cells were incubated for 30 minutes at 4.degree. C. with the
staining mixture, washed two times with PBS and resuspended in 200
.mu.l of PBS. FACS analysis was performed with a 5-laser
LSR-Fortessa and data analysis was conducted using the FlowJo
version 10 software. DCs were identified as viable, single cells
highly positive for CD11c and MHC class II and negative for CD3,
NK1.1 and CD19. CD70 and CD86 expression, both DC activation
markers, was analyzed on DCs three days post therapy injection.
Viable CD45-, CD3- and CD8-positive single cells were identified as
CD8.sup.+ T cells. CD8.sup.+ T cells were analyzed for Ki67 FITC
(eBioscience, clone SolA15, Cat. No. 11-5698-82) expression and
total numbers of CD8.sup.+ T cell in the tumors were determined
using absolute cell count beads (Invitrogen, Cat. No. 01-1234).
[0770] As shown in FIG. 23A, FIG. 23B, and FIG. 23C, on day 3
enzymes suggestive of hepatocellular injury were increased in mice
injected with a single i.p. dose of CD40 but not in mice injected
FAP-CD40 or vehicle alone. In addition, the body weight was
deceased and the spleen weight was increased of CD40-treated mice
three days post treatment compared to mice injected with FAP-CD40
4+1 or vehicle alone (FIG. 23D and FIG. 23E, respectively)
indicating less severe side effects in animals treated with the
FAP-targeted CD40 antibodies compared to animals treated with the
parental CD40 antibody. Although to a lesser extent than FGK4.5,
FAP-targeted anti-CD40 antibodies induced a significant increase in
DC activation (CD86 and CD70 expression) in tumor-draining lymph
nodes three days post treatment (FIG. 24A and FIG. 24B) and
CD8.sup.+ T cell proliferation (Ki68 expression and cell numbers)
in tumors eight days post treatment (FIG. 24C and FIG. 24D)
compared to vehicle-treated mice. In summary, the FAP-targeted
anti-CD40 molecule with FAP-dependent activation of CD40 in a 4+1
format induces potent DC and T cell activation in tumor-bearing
mice with reduced systemic toxicity compared to the untargeted
anti-CD40 parental antibody FGK4.5.
Example 4
Generation and Production of Humanized Variants of Anti-CD40
Antibody S2C6
4.1 First Generation of Humanized Variants of Anti-CD40 Antibody
S2C6
4.1.1 Methodology
[0771] Anti-CD40 antibody S2C6 is disclosed in WO 2000/075348 and
has the VH domain of SEQ ID NO:129 and the VL domain of SEQ ID
NO:130. Variants thereof were created as described in the
following. For the identification of a suitable human acceptor
framework during the humanization of the anti-CD40 binder S2C6, a
combination of two methodologies was used. On the one hand, a
classical approach was taken by searching for an acceptor framework
with high sequence homology, grafting of the CDRs on this
framework, and evaluating which back-mutations can be envisaged.
More explicitly, each amino acid difference of the identified
frameworks to the parental antibody was judged for impact on the
structural integrity of the binder, and back mutations towards the
parental sequence were introduced whenever appropriate. The
structural assessment was based on Fv region homology models of
both the parental antibody and its humanized versions created with
an in-house antibody structure homology modeling tool implemented
using the Biovia Discovery Studio Environment, version 4.5.
[0772] On the other hand, an in-house developed in silico tool was
used to predict the orientation of the VH and VL domains of the
humanized versions towards each other (see WO 2016062734
incorporated herein by reference). The results were compared to the
predicted VH-VL domain orientation of the parental binder to select
for framework combinations which are close in geometry to the
starting antibody. The rational is to detect possible amino acid
exchange in the VH-VL interface region that might lead to
disruptive changes in the pairing of the two domains.
4.1.2 Choice of Acceptor Framework and Adaptations Thereof
[0773] The acceptor framework was chosen as described in Table 12
below:
TABLE-US-00015 TABLE 12 Acceptor framework Choice of human Identity
to human V- Murine V-region acceptor V-region region germline after
germline germline grafting (BLASTp): S2C6 VH IGHV1-26*01 IGHV1-2*01
91.8% S2C6 VL IGKV1-110*01 IGKV2-30*02 92.0%
[0774] Post-CDR3 framework regions were adapted from human IGHJ
germline IGHJ6*01/02 (YYYYYGMDVWGOGTTVTVSS) and human IGKJ germline
IGKJ1*01 (WTFGOGTKVEIK). The part relevant for the acceptor
framework is indicated as underlined.
[0775] Based on structural considerations, back mutations from the
human acceptor framework to the amino acid in the parental binder
were introduced at positions H48 (M>I) and H71 (R>V) of the
VH region and at positions L36 (F>Y), L46 (R>L) and L87
(Y>F) of the VL region (Kabat numbering). Furthermore, two
positions in CDR-H2 were identified as promising candidates for
forward mutations, i.e., amino acid exchanges from parental binder
to human acceptor germline in order to increase overall human
character, namely H60 (N>A) and H64 (K>Q).
[0776] In order to address putative developability hotspots
(asparagine deamidation), further changes with regard to the
parental binder were introduced at positions H52b (N>Q) and H54
(N>A) in VH, and L27f (N>Q), L28 (G>P), L29 (N>Q) and
L30 (T>I) in VL (Kabat numbering).
[0777] In the following Table 13 the VH-VL pairing matrix is
shown:
TABLE-US-00016 hVK_6 hVK_9 hVK_1 hVK_4 hVK_5 bF36Y.sub.-- hVK_7
hVK_8 bF36Y.sub.-- IMGT.sub.-- hVK_3 bF36Y.sub.-- bF36Y.sub.--
bR46L.sub.-- bF36Y.sub.-- bF36Y.sub.-- bR46L.sub.-- hVK_2.sub.--
hVK_2 bF36Y.sub.-- bR46L.sub.-- bR46L.sub.-- bY87F.sub.--
bR46L.sub.-- bR46L.sub.-- bY87F.sub.-- 30_base.sub.-- bF36Y.sub.--
bR46L.sub.-- bY87F.sub.-- bY87F.sub.-- dG28P.sub.-- bY87F.sub.--
bY87F.sub.-- dN27fQ.sub.-- graft bR46L bY87F dG28P dT30I dT30I
dN27fQ dN29Q dN29Q hVH_1 IMGT_hVH_1.sub.-- 2_base_graft hVH_2
bM48I_bR71V x x x x x x x x hVH_3 bM48I_bR71V.sub.-- x x x x x x x
x dN54A hVH_4 bM48I_bR71V.sub.-- x x x x x x x x dN52bQ_dN254A
hVH_5 bM48I_bR71V.sub.-- x x fK64Q hVH_6 bM48I_bR71V.sub.-- x x
fN60A hVH_7 bM48I_bR71V.sub.-- x x fN60A_fK64Q
Back mutations prefixed with b, forward mutations prefixed with f,
and mutations to address developability hotspots prefixed with
d
4.1.3 VH and VL domains of the resulting humanized CD40
antibodies
[0778] The resulting VH domains of humanized CD40 antibodies can be
found in Table 14 below and the resulting VL domains of humanized
CD40 antibodies are listed in Table 15 below.
TABLE-US-00017 TABLE 14 Amino acid sequences of the VH domains of
humanized CD40 antibodies Seq ID Description Sequence No IMGT_hVH_1
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 45
GLEWMGrvipnnggtsynqkfkgRVTSTRDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_2
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 46
GLEWIGrvipnnggtsynqkfkgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_3
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 47
GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_4
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 48
GLEWIGrvipqaggtsynqkfkgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_5
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 49
GLEWIGrvipnnggtsynqkfqgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_6
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 50
GLEWIGrvipnnggtsyaqkfkgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_7
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 51
GLEWIGrvipnnggtsyaqkfqgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_2_N288A
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 52
GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_5_N288A
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 53
GLEWIGrvipnaggtsynqkfqgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_6_N288A
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 54
GLEWIGrvipnaggtsyaqkfkgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS IMGT_hVH_7_N288A
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 55
GLEWIGrvipnaggtsyaqkfqgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSS
TABLE-US-00018 TABLE 15 Amino acid sequences of the VL domains of
humanized CD40 antibodies Seq ID Description Sequence No IMGT_hVK_1
DVVMTQSPLSLPVTLGQPASISCrssqslvhsngntflhWFQQ 56
RPGQSPRRLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYYCsqtthvpwtFGQGTKVEIK IMGT_hVK_2
DVVMTQSPLSLPVTLGQPASISCrssqslvhsngntflhWYQQ 57
RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYYCsqtthvpwtFGQGTKVEIK IMGT_hVK_3
DVVMTQSPLSLPVTLGQPASISCrssqslvhsngntflhWYQQ 58
RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCsqtthvpwtFGQGTKVEIK IMGT_hVK_4
DVVMTQSPLSLPVTLGQPASISCrssqslvhsnpntflhWYQQ 59
RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCsqtthvpwtFGQGTKVEIK IMGT_hVK_5
DVVMTQSPLSLPVTLGQPASISCrssqslvhsngniflhWYQQ 60
RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCsqtthvpwtFGQGTKVEIK IMGT_hVK_6
DVVMTQSPLSLPVTLGQPASISCrssqslvhsnpniflhWYQQ 61
RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCsqtthvpwtFGQGTKVEIK IMGT_hVK_7
DVVMTQSPLSLPVTLGQPASISCrssqslvhsqgntflhWYQQ 62
RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCsqtthvpwtFGQGTKVEIK IMGT_hVK_8
DVVMTQSPLSLPVTLGQPASISCrssqslvhsngqtflhWYQQ 63
RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCsqtthvpwtFGQGTKVEIK IMGT_hVK_9
DVVMTQSPLSLPVTLGQPASISCrssqslvhsqgqtflhWYQQ 64
RPGQSPRLLIYtvsnrfsGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCsqtthvpwtFGQGTKVEIK
[0779] The humanized amino acid sequences for heavy and light chain
variable regions of S2C6 variants were backtranslated in to DNA and
the resulting cNDA were synthesized (GenArt) and then cloned into
heavy chain expression vectors as fusion proteins with human IgG1
backbones/human CH1-Hinge-CH2-CH3 with LALA and PG mutations
(Leucine 234 to Alanine, Leucine 235 to Alanine, Proline 329 to
Glycine) abrogating effector functions or into light chain
expression vectors as fusion proteins to human C-kappa. LC and HC
Plasmids were then cotransfected into HEK293 and purified after 7
days from supernatants by standard methods for antibody
purification.
4.2 Second Generation of Humanized Variants of Anti-CD40 Antibody
S2C6
4.2.1 Methodology
[0780] As for Example 4.1, for the identification of a suitable
human acceptor framework during the humanization of the anti-CD40
binder S2C6 a combination of two methodologies was used. On the one
hand, a classical approach was taken by searching for an acceptor
framework with high sequence homology, grafting of the CDRs on this
framework, and evaluating which back-mutations can be envisaged.
More explicitly, each amino acid difference of the identified
frameworks to the parental antibody was judged for impact on the
structural integrity of the binder, and back mutations towards the
parental sequence were introduced whenever appropriate. The
structural assessment was based on Fv region homology models of
both the parental antibody and its humanized versions created with
an in-house antibody structure homology modeling tool implemented
using the Biovia Discovery Studio Environment, version 4.5.
[0781] On the other hand, an in-house developed in silico tool was
used to predict the orientation of the VH and VL domains of the
humanized versions towards each other (see WO 2016062734
incorporated herein by reference). The results were compared to the
predicted VH-VL domain orientation of the parental binder to select
for framework combinations which are close in geometry to the
starting antibody. The rational is to detect possible amino acid
exchange in the VH-VL interface region that might lead to
disruptive changes in the pairing of the two domains.
4.2.2 Choice of Acceptor Framework and Adaptations Thereof
[0782] Two different acceptor frameworks were chosen as described
in Table 16 and Table 18 below.
TABLE-US-00019 TABLE 16 Acceptor framework 1: "IGHV1-IGKV2D" Choice
of human Identity to human V- Murine V-region acceptor V-region
region germline after germline germline grafting (BLASTp): S2C6 VH
IGHV1-26*01 IGHV1-2*05 91.8% S2C6 VL IGKV1-110*01 IGKV2D-29*02
88.0%
[0783] Post-CDR3 framework regions were adapted from human IGHJ
germline IGHJ6*01/02 (YYYYYGMDVWGQGTTVTVSS) and human IGKJ germline
IGKJ4*01/02 (LTFGGGTKVEIK). The part relevant for the acceptor
framework is indicated in bold script.
[0784] Based on structural considerations, back mutations from the
human acceptor framework to the amino acid in the parental binder
were introduced at positions H43 (Q>K), H44 (G>S), H69
(M>L), H71 (R>V), H73 (T>K), H88 (V>A) and H105
(Q>H) of the VH region and at positions L2 (I>V), L4
(M>V), L87 (Y>F) and L104 (V>L) of the VL region. In one
variant, mutation T70S (VH) was included to study the effect of a
slightly more hydrophilic residue at this position.
[0785] All variants include the N54A mutation (VH) to address a
putative developability hotspot (asparagine deamidation). All
positions are given in the Kabat EU numbering scheme.
[0786] In the following Table 17 the Humanization variant VH-VL
pairing matrix is shown:
TABLE-US-00020 VL1d VL1c bI2V, VL1b bI2V, bM4V, VL1a bM4V, bM4V,
bY783F, bY87F bY87F bY83F bV104L VH1a bG44S, bM69L, bR71V, bT73K,
bV88A x x x x VH1b bQ43K, bG44S, bM69L, bR71V, bT73K, x x x x bV88A
VH1c bG44S, bM69L, bR71V, bT73K, bV88A, x x x x bQ105H VH1d bG44S,
bM69L, bR71V, bT73K, bV88A, x x x x xT70S
Mutation N54A applies to all VH variants and is not explicitly
mentioned. Back mutations prefixed with b, forward mutations
prefixed with f, and other mutations prefixed with x
TABLE-US-00021 TABLE 18 Acceptor framework 2: "IGHV3-IGKV1" Choice
of human Identity to human V- Murine V-region acceptor V-region
region germline after germline germline grafting (BLASTp): S2C6 VH
IGHV1-26*01 IGHV3-23*02 79.6% S2C6 VL IGKV1-110*01 IGKV1-39*01
79.0%
[0787] Post-CDR3 framework regions were adapted from human IGHJ
germline IGHJ6*01/02 (YYYYYGMDVWGQGTTVTVSS) and human IGKJ germline
IGKJ4*01/02 (LTFGGGTKVEIK). The part relevant for the acceptor
framework is indicated in bold script.
[0788] Based on structural considerations, back mutations from the
human acceptor framework to the amino acid in the parental binder
were introduced at positions H44 (G>S), H49 (S>G), H71
(R>V), H78 (L>A), H94 (K>R) and H105 (Q>H) of the VH
region and at positions L42 (K>Q), L43 (A>S) and L87 (Y>F)
of the VL region. Furthermore, four positions in CDR-H2 were
identified as promising candidates for forward mutations, i.e.,
amino acid exchanges from parental binder to human acceptor
germline in order to increase overall human character, namely H60
(N>G), H61 (Q>D), H62 (K>S) and H63 (F>V).
[0789] All variants include the N54A mutation (VH) to address a
putative developability hotspot (asparagine deamidation). All
positions are given in the Kabat EU numbering scheme.
[0790] In the following Table 19 the Humanization variant VH-VL
pairing matrix is shown:
TABLE-US-00022 VL2b bK42Q, VL2a bA43S, bY87F bY87F VH2a bS49G,
bR71V, bL78A, bK94R x x VH2b bG44S, bS49G, bR71V, bL78A, bK94R x x
VH2c bS49G, bR71V, bL78A, bK94R, bQ105H x x VH2d bS49G, fN6OG,
fQ61D, fK62S, fF63V, x x bR71V, bL78A, bK94R
[0791] Back mutations prefixed with b, forward mutations prefixed
with f.
4.2.3 VH and VL Domains of the Resulting Humanized CD40
Antibodies
[0792] The resulting VH and VL domains of humanized CD40 antibodies
based on acceptor framework 1 can be found in Table 17 below and
the resulting VH and VL domains of humanized CD40 antibodies based
on acceptor framework 2 are listed in Table 18 below.
TABLE-US-00023 TABLE 20 Amino acid sequences of the VH and VL
domains of humanized CD40 antibodies based on acceptor framework 1
Seq ID Description Sequence No VH1a
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 171
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSS VH1b
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 172
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSS VH1c
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 173
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGHGTTVTVSS VH1d
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 174
SLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSS VL1a
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 175
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIK VL1b
DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 176
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIK VL1c
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 177
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIK VL1d
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 178
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKLEIK
TABLE-US-00024 TABLE 21 Amino acid sequences of the VH and VL
domains of humanized CD40 antibodies based on acceptor framework 2
Seq ID Description Sequence No VH2a
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 179
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSS VH2b
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 180
SLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSS VH2c
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 181
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGHGTTVTVSS VH2d
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 182
GLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSS VH2ab
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYMHWVRQAPGK 183
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSS VH2ac
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 184
GLEWVGRVIPNAGGTSYNQKVKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSS VL2a
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 185
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIK VL2b
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 186
KPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIK VL2ab
DIQMTQSPSSLSASVGDRVTITCRASQSLVHSNGNTFLHWYQQ 187
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIK VL2ac
DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTFLHWYQQ 188
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIK
4.2.4 New Humanized CD40 Antibodies in huIgG1_LALA_PG Format
[0793] Based on the new humanization variants of VH and VL new CD40
antibodies were expressed as huIgG1 antibodies with an effector
silent Fc (P329G; L234, L235A) to abrogate binding to Fc.gamma.
receptors according to the method described in WO 2012/130831
A1.
TABLE-US-00025 TABLE 22 Nomenclature for VH/VL combinations
expressed as huIgG1_LALA_PG antibodies VL1a VL1b VL1c VL1d VL2a
VL2b VL2ab VL2ac VH1a P1AE P1AE P1AE P1AE 0817 1001 0993 0996 VH1b
P1AE P1AE P1AE P1AE 1002 1003 1004 1005 VH1c P1AE P1AE P1AE P1AE
0997 1006 0818 0998 VH1d P1AE P1AE P1AE P1AE 0999 1007 1000 0819
VH2a P1AE P1AE 0400 0404 VH2b P1AE P1AE 0401 0405 VH2c P1AE P1AE
0402 0406 VH2d P1AE P1AE 0403 0407 VH2ab P1AE P1AE 1125 1126 VH2ac
P1AE P1AE 1134 1135
[0794] The full-length sequences of humanized CD40 antibodies as
human IgG1_LALAPG antibodies can be found in Table 20.
TABLE-US-00026 TABLE 23 Amino acid sequences of the humanized CD40
IgG1_LALAPG antibodies Seq ID Antibody Sequence No P1AE0400
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 189 heavy chain
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0400
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 190 light chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0401
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 191 heavy chain
SLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0401
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 192 light chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0402
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 193 heavy chain
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0402
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 194 light chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0403
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 195 heavy chain
GLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0403
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 196 light chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0404
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 197 heavy chain
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0404
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 198 light chain
KPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0405
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 199 heavy chain
SLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0405
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 200 light chain
KPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0406
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 201 heavy chain
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0406
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 202 light chain
KPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0407
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 203 heavy chain
GLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0407
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 204 light chain
KPGQSPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0816
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 259 heavy chain
SLEWMGRVIPNNGGTSYNQKFQGRVTISVDKSISTAYMELSSL (control)
RSEDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0816
DVVVTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTFLHWYLQ 260 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (control)
DVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0817
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 205 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0817
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 206 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0818
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 207 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0818
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 208 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0819
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 209 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0819
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 210 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0993
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 211 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0993
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 212 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0996
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 213 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0996
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 214 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0997
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 215 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0997
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 216 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0998
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 217 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0998
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 218 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE0999
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 219 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE0999
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 220 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1000
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 221 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1000
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 222 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1001
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 223 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1001
DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 224 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1002
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 225 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1002
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 226 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1003
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 227 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1003
DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 228 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1004
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 229 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1004
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 230 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1005
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGK 231 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1005
DVVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 232 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1006
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 233 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGHGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1006
DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 234 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1007
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 235 heavy chain
SLEWMGRVIPNAGGTSYNQKFKGRVTLSVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1007
DIVVTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 236 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1125
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYMHWVRQAPGK 237 heavy chain
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1125
DIQMTQSPSSLSASVGDRVTITCRASQSLVHSNGNTFLHWYQQ 238 light chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC
P1AE1126 EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYMHWVRQAPGK 239 heavy
chain GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1126
DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTFLHWYQQ 240 light chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC P1AE1135
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 241 heavy chain
GLEWVGRVIPNAGGTSYNQKVKGRFTISVDNSKNTAYLQMNSL
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE1135
DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTFLHWYQQ 242 light chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC
4.2.5 Production of the New Humanized CD40 Antibodies in
huIgG1_LALA_PG Format
[0795] The antibodies were expressed by transient transfection of
HEK293-F cells grown in suspension with expression vectors encoding
the different peptide chains. Transfection into HEK293-F cells
(Invitrogen, USA) was performed according to the cell supplier's
instructions using Maxiprep (Qiagen, Germany) preparations of the
antibody vectors, F17 based medium (Invitrogen, USA), PEIpro
(Polyscience Europe GmbH) and an initial cell density of 1-2
million viable cells/ml in serum free FreeStyle 293 expression
medium (Invitrogen). Cell culture supernatants were harvested after
7 days of cultivation in shake flasks or stirred fermenters by
centrifugation at 14000 g for 30 minutes and filtered through a
0.22 .mu.m filter.
[0796] The antibodies were purified from cell culture supernatants
by affinity chromatography using MabSelectSure-Sepharose .TM. (GE
Healthcare, Sweden) chromatography. Briefly, sterile filtered cell
culture supernatants were captured on a MabSelect SuRe resin
equilibrated with PBS buffer (10 mM Na.sub.2HPO.sub.4, 1 mM
KH.sub.2PO.sub.4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with
equilibration buffer and eluted with 25 mM citrate, pH 3.0. After
neutralization with 1 M Tris pH 9.0, aggregated protein was
separated from monomeric antibody species by size exclusion
chromatography (Superdex 200, GE Healthcare) in 20 mM histidine,
140 mM NaCl, pH 6.0. Monomeric protein fractions were pooled,
concentrated if required using e.g. a MILLIPORE Amicon Ultra (30KD
MWCO) centrifugal concentrator and stored at -80.degree. C. Sample
aliquots were used for subsequent analytical characterization e.g.
by CE-SDS, size exclusion chromatography, mass spectrometry and
endotoxin determination.
[0797] The production yield for the different humanized CD40
antibodies is shown in Table 21 as titer values calculated from the
yield after preparative affinity chromatography using
MabSelectSure-Sepharose.TM. chromatography.
[0798] Purity and molecular weight of the molecule after the final
purification step were analyzed by CE-SDS analyses in the presence
and absence of a reducing agent. The Caliper LabChip GXII system
(Caliper Lifescience) was used according to the manufacturer's
instruction.
[0799] The aggregate content of the molecule was analyzed using a
TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25
mM potassium phosphate, 125 mM sodium chloride, 200 mM L-arginine
monohydrocloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at
25.degree. C.
[0800] For direct comparison of all antibodies the thermal
stability was monitored by Static Light Scattering (SLS) and by
measuring the intrinsic protein fluorescence in response to applied
temperature stress. 30 .mu.g of filtered protein sample with a
protein concentration of 1 mg/ml was applied in duplicate to an
Optim 2 instrument (Avacta Analytical Ltd). The temperature was
ramped from 25 to 85.degree. C. at 0.1.degree. C./min, with the
radius and total scattering intensity being collected. For
determination of intrinsic protein fluorescence the sample was
excited at 266 nm and emission was collected between 275 nm and 460
nm. For all antibodies the aggregation temperature (Tagg) was
between 64 and 69.degree. C. and is provided in Table 24 or Table
25 below.
[0801] The production yield for the humanized CD40 antibodies with
the different frameworks is shown in Table 24 or Table 25
below.
TABLE-US-00027 TABLE 24 Production titer, humanness and aggregation
temperature of humanized CD40 antibodies based on acceptor
framework 2 Titer humanness Antibody VH/VL [.mu.g/ml] (VH/VL in %)
Tagg P1AD4470 control 140 77.6/78 68 P1AE0400 VL2a/VH2a 219 77.6/78
69 P1AE0401 VL2a/VH2b 162 76.5/78 69 P1AE0402 VL2a/VH2c 196 77.6/78
69 P1AE0403 VL2a/VH2d 137 80.6/78 67 P1AE0404 VL2b/VH2a 165 77.6/76
69 P1AE0405 VL2b/VH2b 128 76.5/76 69 P1AE0406 VL2b/VH2c 154 77.6/76
69 P1AE0407 VL2b/VH2d 102 80.6/76 67
TABLE-US-00028 TABLE 25 Production titer, humanness and aggregation
temperature of humanized CD40 antibodies based on acceptor
framework 1 Titer humanness Antibody VH/VL [.mu.g/ml] (VH/VL in %)
Tagg P1AE0816 control 8.5 84.7/84 64 P1AE0817 VH1a/VL1a 62 86.7/87
67 P1AE0818 VH1c/VL1c 47 86.7/85 66 P1AE0819 VH1d/VL1d 90 85.7/85
67 P1AE0993 VH1a/VL1c 34 86.7/85 67 P1AE0996 VH1a/VL1d 16 86.7/85
67 P1AE0997 VH1c/VL1a 44 86.7/87 66 P1AE0998 VH1c/VL1d 24 86.7/85
66 P1AE0999 VH1d/VL1a 34 85.7/87 67 P1AE1000 VH1d/VL1c 16 85.7/85
66 P1AE1001 VH1a/VL1b 34 86.7/86 65 P1AE1002 VH1b/VL1a 46 85.7/87
67 P1AE1003 VH1b/VL1b 49 85.7/86 66 P1AE1004 VH1b/VL1c 60 85.7/85
67 P1AE1005 VH1b/VL1d 7 85.7/85 65 P1AE1006 VH1c/VL1b 24 86.7/86 65
P1AE1007 VH1d/VL1b 34 85.7/86 67
4.2.6 Generation of Recombinant Human and Cynomolgus Monkey CD40
Extracellular Domain Protein
[0802] Following constructs were cloned and expressed by transient
expression in HEK293 cells:
[0803] 1) Human CD40 extracellular domain (amino acids 21-193 of
SEQ ID NO:1, NCBI accession number NP_001241) with C-terminal
His-AviTag.TM. tag (SEQ ID NO:266)
[0804] 2) Cynomolgus monkey (macaca fascicularis) CD40
extracellular domain (amino acids 21-193, cynomolgus CD40
extracellular domain sequence was taken from Roche cynomolgus cDNA
database, unpublished data) with C-terminal His-AviTag.TM. tag (SEQ
ID NO:267)
[0805] CD40 extracellular domain antigens for binding analysis were
generated by gene synthesis (Eurofins Genomics GmbH service,
Germany), cloned via unique restriction sites into Roche's in house
expression vector using standard cloning procedures. Cloning of all
constructs was verified by sequencing. All antigens were expressed
under the control of the CMV-promoter. For transient expression of
the CD40 extracellular domain constructs, suspension-adapted
HEK293-F cells (Life Technologies, USA) were transfected with the
respective plasmids: In general, 1L of HEK293-F cells at about
2.times.10.sup.6 cells/ml were transfected with a total of 500
.mu.g plasmid DNA complexed by the PEIpro Transfection Reagent
(Polysciences Europe GmbH, Germany) according to manufacturer's
instructions. After transfection, HEK293-F cells were incubated for
6 days. The cells were subsequently harvested by centrifugation and
the protein-containing supernatant was filtered using a 0.22 .mu.m
vacuum filtration system (Millipore). The His-AviTag.TM. tagged
proteins were purified by IMAC affinity chromatography using
complete-His-Tag resin (Roche Diagnostics). After washing with 50
mM Na.sub.2PO.sub.4, 300 mM NaCl, pH 8.0, His-AviTag.TM. fusion
proteins were eluted using washing buffer supplemented with 500 mM
Imidazol at pH 7.0. Aggregated protein was separated from monomeric
fusion proteins by size exclusion chromatography (Superdex 75, GE
Healthcare) in 20 mM Tris, 150 mM NaCl, pH 7.4. Monomeric protein
fractions were pooled, concentrated if required using e.g. a
MILLIPORE Amicon Ultra (10KD MWCO) centrifugal concentrator and
stored at -80.degree. C. Sample aliquots were used for subsequent
analytical characterization e.g. by CE-SDS, size exclusion
chromatography and mass spectrometry.
[0806] Biotinylation of CD40 Extracellular Domain:
[0807] Enzymatic site specific biotinylation of human or cynomolgus
CD40 extracellular domain constructs containing a C-terminal
AviTag.TM. was performed by using the BirA biotin-protein ligase
kit (Avidity LLC, USA) according to manufactures instruction.
Briefly, 1/10 volume of BiomixA (10.times. concentration: 0.5M
bicine buffer, pH 8.3) and BiomixB (10.times. concentration: 100 mM
ATP, 100 mM MgOAc, 500 .mu.M d-biotin) was added to AviTag.TM.
containing protein followed by addition of 2.5 .mu.g BirA ligase
per 10 nmol protein. The reaction mixture was incubated at
30.degree. C. for 1 h and purified by size exclusion chromatography
on a Superdex75 prep grade prepacked HiLoad column (GE Healthcare,
Sweden).
4.2.7 Human/Cynomolgus CD40 Binding Surface Plasmon Resonance
Spectroscopy Assay
[0808] Around 12000 resonance units (RU) of the capturing system
(10 .mu.g/ml goat anti human F(ab)'.sub.2; Order Code: 28958325; GE
Healthcare Bio-Sciences AB, Sweden) were coupled on a CM5 chip (GE
Healthcare BR-1005-30) at pH 5.0 by using an amine coupling kit
supplied by the GE Healthcare. The sample and system buffer was
PBS-T (10 mM phosphate buffered saline including 0.05% Tween20) pH
7.4. The flow cell was set to 25.degree. C.--and the sample block
set to 12.degree. C.--and primed with running buffer twice. The
antibody was captured by injecting a 50 nM solution for 30 sec at a
flow of 5 .mu.l/min. Association was measured by injection of human
CD40 extra cellular domain or cynomolgus monkey CD40 extracellular
domain in various concentrations in solution for 300 sec at a flow
of 30 .mu.l/min starting with 300 nM in 1:3 dilutions. The
dissociation phase was monitored for up to 1200 sec and triggered
by switching from the sample solution to running buffer. The
surface was regenerated by 60 sec washing with a Glycine pH 2.1
solution at a flow rate of 30 .mu.l/min. Bulk refractive index
differences were corrected by subtracting the response obtained
from a goat anti human F(ab').sub.2 surface. Blank injections are
also subtracted (=double referencing). For calculation of apparent
K.sub.D and other kinetic parameters the Langmuir 1:1 model was
used. The apparent Kd was calculated using the Biacore.TM. B4000
evaluation software (version 1.1).
4.2.8 Cellular Binding Assay for Characterisation of CD40-Specific
Humanized Antibodies
[0809] CD40 positive cells (Raji cells) were detached from the
culture bottle using Trypsin and were counted using a Casy cell
counter. After pelleting at 4.degree. C., the cells were
resuspended in FACS Buffer (2.5% FCS in PBS), adjusted to 2.0E+06
cells/mL, and dispensed to 96-well PP V-bottom-plates (25
.mu.L/well=5.0E+04Zellen/well).
[0810] The CD40 specific antibodies were adjusted to 20 .mu.g/mL in
FACS buffer, resulting in a final concentration of 10 .mu.g/mL. 20
.mu.l were added to 25 .mu.l cell suspension and incubated for 1 h
at 4.degree. C. The cells were then washed twice in FACS buffer.
After washing, the cells were resuspended in 50 .mu.L FACS-buffer
containing secondary antibody (<huIgG>-Alexa488, c=10
.mu.g/mL) and incubated 1 h bei 4.degree. C. The cells were then
washed twice in FACS buffer and resuspended in 70 .mu.l/well FACS
buffer for measurement using a FACS Canto (BD, Pharmingen).
[0811] In Table 26 the affinity of the humanized CD40 antibodies
(measured by Biacore) and the cellular binding to CD40 expressing
cells (Raji cells) is shown.
TABLE-US-00029 TABLE 26 Affinity and cellular binding of humanized
CD40 antibodies to CD40 expressing cells EC.sub.50 [.mu.g/ml]
cellular Affinity binding ID VH/VL [nM] Ka (1/Ms) Kd (1/s) (Raji)
P1AD4470 control 4.6 1.69E+06 7.81E-03 0.09 P1AE0400 VL2a/VH2a 4.2
1.68E+06 6.99E-03 0.12 P1AE0401 VL2a/VH2b 4.6 1.69E+06 7.87E-03
0.13 P1AE0402 VL2a/VH2c 4.2 1.67E+06 7.09E-03 0.13 P1AE0403
VL2a/VH2d 29 1.40E+06 4.07E-02 0.12 P1AE0404 VL2b/VH2a 4.2 1.63E+06
6.93E-03 0.11 P1AE0405 VL2b/VH2b 5.1 1.61E+06 8.14E-03 0.09
P1AE0406 VL2b/VH2c 4.2 1.67E+06 7.09E-03 0.09 P1AE0407 VL2b/VH2d 30
1.19E+06 3.55E-02 0.12 P1AE0816 control 8.7 2.53E+06 2.19E-02 0.09
P1AE0817 VH1a/VL1a 2.5 2.40E+06 5.93E-03 0.09 P1AE0818 VH1c/VL1c
3.2 2.63E+06 8.47E-03 0.14 P1AE0819 VH1d/VL1d 3.4 2.59E+06 8.77E-03
0.11 P1AE0993 VH1a/VL1c 3.4 2.68E+06 8.98E-03 0.13 P1AE0996
VH1a/VL1d 3.5 2.59E+06 9.08E-03 0.12 P1AE0997 VH1c/VL1a 2.3
2.59E+06 6.03E-03 0.12 P1AE0998 VH1c/VL1d 3.3 2.70E+06 8.96E-03
0.12 P1AE0999 VH1d/VL1a 2.4 2.45E+06 5.92E-03 0.15 P1AE1000
VH1d/VL1c 3.2 2.68E+06 8.62E-03 0.14 P1AE1001 VH1a/VL1b 2.7
2.56E+06 6.81E-03 0.08 P1AE1002 VH1b/VL1a 2.2 2.54E+06 5.57E-03
0.13 P1AE1003 VH1b/VL1b 2.5 2.46E+06 6.06E-03 0.13 P1AE1004
VH1b/VL1c 3 2.63E+06 7.95E-03 0.14 P1AE1005 VH1b/VL1d 3.2 2.58E+06
8.16E-03 0.11 P1AE1006 VH1c/VL1b 2.6 2.53E+06 6.51E-03 0.14
P1AE1007 VH1d/VL1b 2.7 2.50E+06 6.62E-03 0.12
4.2.9 Antibody Characterisation by UHR-ESI-QTOF Mass
Spectrometry
[0812] The samples were desalted by HPLC on a Sephadex G25
5.times.250 mm column (Amersham Biosciences, Freiburg, Germany)
using 40% acetonitrile with 2% formic acid (v/v). The total mass
was determined by UHR-ESI-QTOF MS on a maXis 4G UHR-QTOF MS system
(Bruker Daltonik, Bremen, Germany) equipped with a TriVersa
NanoMate source (Advion, Ithaca, N.Y.). Data acquisition was done
at 900-4000 m/z (ISCID: 0.0 eV). The raw mass spectra were
evaluated and transformed into individual relative molar masses
using an in-house developed software tool.
4.2.10 Thermal Stability Evaluation of Antibodies
[0813] Samples are prepared at a concentration of 1 mg/mL in 20 mM
Histidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into
an optical 384-well plate by centrifugation through a 0.4 .mu.m
filter plate and covered with paraffine oil. The hydrodynamic
radius is measured repeatedly by dynamic light scattering on a
DynaPro Plate Reader (Wyatt) while the samples are heated with a
rate of 0.05.degree. C./min from 25 .degree. C. to 80 .degree. C.
Alternatively, samples were transferred into a 10 .mu.L
micro-cuvette array and static light scattering data as well as
fluorescence data upon excitation with a 266 nm laser were recorded
with an Optim1000 instrument (Avacta Inc.), while they were heated
at a rate of 0.1.degree. C./min from 25.degree. C. to 90.degree. C.
The aggregation onset temperature is defined as the temperature at
which the hydrodynamic radius (DLS) or the scattered light
intensity (Optim1000) starts to increase. The melting temperature
is defined as the inflection point in a graph showing fluorescence
intensity vs. wavelength.
Example 5
Generation and Production of Bispecific Constructs with New
Humanized CD40 Antibody Variants
5.1 Generation of Bispecific Antigen Binding Molecules Targeting
CD40 and Fibroblast Activation Protein (FAP)
[0814] The cDNAs encoding a VH domain and a VL domain as described
in Example 4 were cloned in frame with the corresponding constant
heavy or light chains of human IgG1 in suitable expression
plasmids. Expression of heavy and light chain is driven by a
chimeric MPSV promoter consisting of the MPSV core promoter and a
CMV enhancer element. The expression cassette also contains a
synthetic polyA signal at the 3' end of the cDNAs. In addition the
plasmid vectors harbor an origin of replication (EBV OriP) for
episomal maintenance of the plasmids.
[0815] In analogy to Example 1, different bispecific CD40-FAP
antibodies are prepared in 4+1 format consisting of four CD40
binding moieties combined with one FAP binding moiety at the
C-terminus of an Fc (FIG. 1A) or in 2+1 and 2+2 formats consisting
of two CD40 binding moieties combined with either one FAP binding
moiety at the C-terminus of an Fc (FIG. 1C and FIG. 1E) or two FAP
binding moieties at the C-terminus of an Fc (FIG. 1D). In addition,
a bispecific antibody consisting of one CD40 binding moiety
combined with one FAP binding moiety is prepared (FIG. 1F). The
generation and preparation of FAP binders 28H1 and 4B9 is described
in WO 2012/020006 A2, which is incorporated herein by reference. To
generate the 4+1 and the 2+1 molecules the knob-into-hole
technology is used to achieve heterodimerization. The S354C/T366W
mutations are introduced in the first heavy chain HC1 (Fc knob
heavy chain) and the Y349C/T366S/L368A/Y407V mutations are
introduced in the second heavy chain HC2 (Fc hole heavy chain). In
the 2+2 molecule the CrossMab technology as described in WO
2010/145792 A1 ensures correct light chain pairing. Independent of
the bispecific format, in all cases an effector silent Fc (P329G;
L234, 234A) is used to abrogate binding to Fc.gamma. receptors
according to the method described in WO 2012/130831 A1. Sequences
of the bispecific molecules are shown in Table 27.
[0816] All genes are transiently expressed under control of a
chimeric MPSV promoter consisting of the MPSV core promoter
combined with the CMV promoter enhancer fragment. The expression
vector also contains the oriP region for episomal replication in
EBNA (Epstein Barr Virus Nuclear Antigen) containing host
cells.
TABLE-US-00030 TABLE 27 Amino acid sequences of the bispecific
antigen binding molecules Seq ID Construct Sequence No CD40
(hVH3/hVK2) .times. FAP (4B9) (4 + 1) with C-terminal VH/VL
hVH3_CD40 QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 131
VHCH1-VHCH1- GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL
Fcknob_PGLALA- RSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS 4B9 VH
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFT
GYYIHWVRQAPGQGLEWIGRVIPNAGGTSYNQKFKGRVTSTVD
TSISTAYMELSRLRSDDTVVYYCAREGIYWWGQGTTVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA
PGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQM
NSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS hVH3_CD40
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 132 VHCH1-
GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL VHCH1-Fchole_PGLALA-
RSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS 4B9 VL
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFT
GYYIHWVRQAPGQGLEWIGRVIPNAGGTSYNQKFKGRVTSTVD
TSISTAYMELSRLRSDDTVVYYCAREGIYWWGQGTTVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQK
PGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPED
FAVYYCQQGIMLPPTFGQGTKVEIK hVK2_CD40
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 133 light chain
RPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (hVH3/hVK2)
.times. FAP (4B9) (2 + 1) with C-terminal VH/VL hVH3_CD40
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 134
VHCH1-Fcknob_PGLALA- GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL
4B9 VH RSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGG
LVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIG
SGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKGWFGGFNYWGQGTLVTVSS
hVH3_CD40 QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 135
VHCH1-Fchole_PGLALA- GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL
4B9 VL RSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGT
LSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGS
RRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLP PTFGQGTKVEIK hVK2_CD40
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 133 light chain
RPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC CD40 (hVH3/hVK2)
.times. FAP (4B9) (2 + 2) hVH3_CD40-
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 136 Fc_PGLALA_4B9_VLCH1
GLEWIGRVIPNAGGTSYNQKFKGRVTSTVDTSISTAYMELSRL (charged)
RSDDTVVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGT
LSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGS
RRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLP
PTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSC hVK2_CD40 LC
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 137 (charged)
RPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 4B9 VHCL
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 138
GLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC CD40
(hVH3/hVK2) .times. FAP (4B9) (2 + 1) with C-terminal crossfab
hVH3_CD40-Fcknob_PGLALA QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ
139 C- GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRL
term_x4B9_FAP_VL_CH1 RSDDTVVYYCARegiywWGQGTTVTVSSASTKGPSVFPLAPSS
(charged) KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGT
LSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGS
RRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLP
PTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSC hVH3_CD40-Fchole_PGLALA
QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ 140 (charged)
GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG
+hVK2_CD40 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 137 LC
(charged) RPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC +4B9 VHCL
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 138
GLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC CD40
(hVH3/hVK2) .times. FAP (4B9) (1 + 1) 4B9-Fcknob_PGLALA
EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQ 141
APRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV
YYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTL
PPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG
hVH3_CD40-Fchole_PGLALA QVQLVQSGAEVKKPGASVKVSCKASgysftgyyihWVRQAPGQ
140 (charged) GLEWIGrvipnaggtsynqkfkgRVTSTVDTSISTAYMELSRL
RSDDTVVYYCARegiywWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG
+hVK2_CD40 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTFLHWYQQ 137 LC
(charged) RPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYYCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC +4B9 VHCL
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 138
GLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC
[0817] Further bispecific antibodies were prepared with the new
humanized CD40 antibody variants as described in Example 4.2.
Specific sequences of such bispecific antibodies are shown in Table
28 below. In particular, different bispecific CD40-FAP antibodies
were prepared in 4+1 format consisting of four CD40 binding
moieties combined with one FAP binding moiety as crossover fab
fragment fused to the C-terminus of the Fc knob chain (FIG. 15F and
FIG. 15G) or in 2+1 format consisting of two CD40 binding moieties
combined with either one FAP binding moiety as crossover fab
fragment, wherein the VL-CH1 chain is fused at the C-terminus of
the Fc knob chain (FIG. 15H) or one FAP binding moiety as crossover
fab fragment, wherein the VH-CL chain is fused at the C-terminus of
the Fc knob chain (FIG. 15I).
TABLE-US-00031 TABLE 28 Amino acid sequences of bispecific antigen
binding molecules Seq ID Construct Sequence No P1AE0889 CD40
(VH2a/VL2a) .times. FAP (28H1) (4 + 1) C-terminal crossfab fusion
28H1 light chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162
cross VHCL GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC VL2a (CD40)
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 243 light chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE (charged)
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH2a (CD40)
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 244 (VHCH1
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL charged_VH2a
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (CD40) (VHCH1
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
charged)-Fcknob_PGLALA_28H1
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK (VLCH1)
SCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVGRVIPNAGGTSYNQKFKGRFTISVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQK
PGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPED
FAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C VH2a (CD40)
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 245 (VHCH1
GLEWVGRVIPNAGGTSYNQKFKGRFTISVDNSKNTAYLQMNSL charged_VH2a
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (CD40) (VHCH1
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ charged)-Fchole_PGLALA
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVGRVIPNAGGTSYNQKFKGRFTISVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPG P1AE0890 CD40 (VH2d/VL2a) .times. FAP
(28H1) (4 + 1) C-terminal crossfab fusion 28H1 light chain
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCL
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC VL2a (CD40)
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQ 243 light chain
KPGKAPKLLIYTVSNRFSGVPSRFSGSGSGTDFTLTISSLQPE (charged)
DFATYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH2d (CD40)
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 246 (VHCH1
GLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSL charged_VH2d
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (CD40) (VHCH1
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
charged)-Fcknob_PGLALA_28H1
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK (VLCH1)
SCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVGRVIPNAGGTSYGDSVKGRFTISVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQK
PGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPED
FAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C VH2d (CD40)
EVQLLESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGK 247 (VHCH1
GLEWVGRVIPNAGGTSYGDSVKGRFTISVDNSKNTAYLQMNSL charged_VH2d
RAEDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (CD40) (VHCH1
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ charged)-Fchole_PGLALA
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGYSFT
GYYIHWVRQAPGKGLEWVGRVIPNAGGTSYGDSVKGRFTISVD
NSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTTVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPG P1AE2024 CD40 (VH1a/VL1a) .times. FAP
(28H1) (4 + 1) C-terminal crossfab fusion 28H1 light chain
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCL
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC VL1a (CD40)
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 248 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (charged)
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 249 (VHCH1)_VH1a
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (CD40) (VHCH1)
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS Fcknob_PGLALA_28H1
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ (VLCH1)
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK (charged)
SCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFT
GYYIHWVRQAPGQSLEWMGRVIPNAGGTSYNQKFKGRVTLTVD
KSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGG
GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQK
PGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPED
FAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 250 (VHCH1)_VH1a
SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (CD40)
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (VHCH1)-Fchole_PGLALA
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ (charged)
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFT
GYYIHWVRQAPGQSLEWMGRVIPNAGGTSYNQKFKGRVTLTVD
KSISTAYMELSRLRSDDTAVYYCAREGIYWWGQGTTVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK
TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPG P1AE2302 CD40 (VH1a/VL1a) .times. FAP
(28H1) (2 + 1) C-terminal crossfab fusion 28H1 light chain
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK 162 cross VHCL
GLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLR
AEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC VL1a (CD40)
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 248 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (charged)
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 251 (VHCH1)
Fcknob_PGLALA_28H1 SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
(VLCH1) RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (charged)
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGT
LSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGAS
TRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIP
PTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSC VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 252 (VHCH1)
Fchole_PGLALA SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (charged)
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE2402
CD40 (VH1a/VL1a) .times. FAP (4B9) (2 + 1) C-terminal crossfab
fusion 4B9 light chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK
138 cross VHCL GLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC VL1a (CD40)
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 248 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (charged)
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 253 (VHCH1)
Fcknob_PGLALA_4B9 SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
(VLCH1) RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (charged)
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGT
LSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGS
RRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLP
PTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSC VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 252 (VHCH1)
Fchole_PGLALA SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (charged)
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE2408
CD40 (VH1a/VL1a) .times. FAP (4B9) (2 + 1) C-terminal crossfab
fusion 4B9 light chain EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQ
254 cross VLCH1 APRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV
YYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC VL1a (CD40)
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 248 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE (charged)
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 255 (VHCH1)
Fcknob_PGLALA_4B9 SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
(VHCL) RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS (charged)
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGG
LVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIG
SGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 252 (VHCH1)
Fchole_PGLALA SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL (charged)
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG P1AE2487
CD40 (VH1a/VL1a) .times. FAP (4B9) (2 + 1) C-terminal crossfab
fusion 4B9 light chain EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQ
254 cross VLCH APRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV
YYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC VL1a (CD40)
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLHWYLQ 256 light chain
KPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYFCSQTTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 257 (VHCH1)
Fcknob_PGLALA_4B9 SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
(VHCL) RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGG
LVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIG
SGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
AKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VH1a (CD40)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQ 258 (VHCH1)
Fchole_PGLALA SLEWMGRVIPNAGGTSYNQKFKGRVTLTVDKSISTAYMELSRL
RSDDTAVYYCAREGIYWWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG
5.2 Production of Bispecific Antigen Binding Molecules Targeting
CD40 and Fibroblast Activation Protein (FAP)
[0818] The bispecific antigen binding molecules targeting CD40 and
fibroblast activation protein (FAP) were expressed by transient
transfection of HEK cells grown in suspension with expression
vectors encoding the 4 different peptide chains. Transfection into
HEK293-F cells (Invitrogen) was performed according to the cell
supplier's instructions using Maxiprep (Qiagen) preparations of the
antibody vectors, F17 medium (Invitrogen, USA), Peipro (Polyscience
Europe GmbH) and an initial cell density of 1-2 million viable
cells/ml in serum free FreeStyle 293 expression medium
(Invitrogen). Cell culture supernatants were harvested after 7 days
of cultivation in shake flasks or stirred fermenters by
centrifugation at 14000 g for 30 minutes and filtered through a
0.22 .mu.m filter.
[0819] The bispecific antibodies were purified from cell culture
supernatants by affinity chromatography using
MabSelectSure-Sepharose.TM. (GE Healthcare, Sweden) chromatography.
Briefly, sterile filtered cell culture supernatants were captured
on a MabSelect SuRe resin equilibrated with PBS buffer (10 mM
Na.sub.2HPO.sub.4, 1 mM KH.sub.2PO.sub.4, 137 mM NaCl and 2.7 mM
KCl, pH 7.4), washed with equilibration buffer and eluted with 25
mM cirate, pH 3.0. After neutralization with 1 M Tris pH 9.0,
aggregated protein was separated from monomeric antibody species by
size exclusion chromatography (Superdex 200, GE Healthcare) in 20
mM histidine, 140 mM NaCl, pH 6.0. Monomeric protein fractions were
pooled, concentrated if required using e.g. a MILLIPORE Amicon
Ultra (30KD MWCO) centrifugal concentrator and stored at
-80.degree. C. Sample aliquots were used for subsequent analytical
characterization e.g. by CE-SDS, size exclusion chromatography,
mass spectrometry and endotoxin determination.
TABLE-US-00032 TABLE 29 Production yield and quality of bispecific
CD40 antigen binding molecules Yield [mg/L] after Protein Purity
(by Affinity to A and SEC % CE- Purity (by human Construct
purification SDS) % SEC) FAP [nM] P1AE2024 CD40 6.6 mg/L 98.2 97.8
1.2 (VH1a/VL1a) .times. FAP (28H1) (4 + 1) C-terminal crossfab
(knob_VL_CH1) P1AE2302 CD40 12 mg/L 99 99.7 0.3 (VH1a/VL1a) .times.
FAP (28H1) (2 + 1) C-terminal crossfab (knob_VL_CH1) P1AE2402 CD40
21 mg/L 96.4 99.2 17.3 (VH1a/VL1a) .times. FAP (4B9) (2 + 1)
C-terminal crossfab (knob_VL_CH1) P1AE2408 CD40 18 mg/L 91.3 95.2
15.3 (VH1a/VL1a) .times. FAP (4B9) (2 + 1) C-terminal crossfab
(knob_VH_Ck) P1AE0408 CD40 .times. FAP 42 mg/L 97.8 96.2 1.5 (28H1)
(2 + 1) head-to-tail P1AE0637 CD40 .times. FAP 9.7 mg/L 98.7 100
0.1 (28H1) (4 + 1) (knob_VL_CH1) P1AE0889 CD40 17 mg/L 96.4 99 nd
(VH2a/VL2a) .times. FAP (28H1) 4 + 1 with C-terminal crossfab
P1AE2487 CD40 nd nd 99.2 nd (VH1a/VL1a) .times. FAP (4B9) (2 + 1)
C-terminal crossfab (knob_VH_Ck)
5.3 Characterization of the Bispecific Antibodies Comprising
Humanized CD40 Antibody Variants and FAP
5.3.1 Binding to Human or Mouse FAP-Expressing Murine Fibroblast
Cells
[0820] The binding to cell surface FAP was tested using human
fibroblast activating protein (huFAP) expressing cells
NIH/3T3-huFAP clone 19 or mouse fibroblast activating protein
(mFAP) expressing cells NIH/3T3-mFAP clone 26 was tested as
described in Example 1.4.1. EC.sub.50 values as measured for some
of the bispecific antigen binding molecules comprising humanized
CD40 antibody variants are shown in Table 7.
5.3.2 Binding to FAP (Surface Plasmon Resonance)
[0821] The capacity of the bispecific constructs to bind human FAP
was assessed by surface plasmon resonance (SPR). All SPR
experiments were performed on a Biacore T200 (Biacore) at 25
.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4,
0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, (Biacore).
[0822] His-tagged human dimeric FAP (recombinant FAP_ECD) was
captured on a CMS chip (GE Healthcare) immobilized with anti-His
antibody (Qiagen Cat. No. 34660) by injection of 500 nM huFAP for
60 s at a flow rate of 10 uL/min, 10 nM murine FAP for 20 s at a
flow rate of 20 uL/min and 10 nM cynoFAP for 20s at a flow rate of
20 uL/min. Immobilization levels for the anti-His antibody of up to
18000 resonance units (RU) were used. Following the capture step,
the bispecific antibodies as well as control molecules were
immediately passed over the chip surface at a concentration ranging
from 0.78-100 nM with a flow rate of 30 .mu.L/minute for 280 s and
a dissociation phase of 180 s. Bulk refractive index differences
were corrected for by subtracting the response obtained in a
reference flow cell, where no FAP was immobilized. Avidity was
determined using the Langmuir 1:1 curve fitting. For bivalent
binding the same 1:1 fitting was used leading to an apparent KD
value.
TABLE-US-00033 TABLE 30 Binding of exemplary bispecific CD40
.times. FAP antigen binding molecules to recombinant human FAP_ECD
(Biacore) KD * Ligand (Avidity) ka (1/Ms) kd (1/s) Control 4B9 IgG1
0.08 nM 2.19E+06 1.72E-04 P1AD9139 4 + 1 with C-terminal 2.7 nM
5.76E+05 1.55E-03 VH/VL fusion P1AE0192 1 + 1 crossMab 2.2 nM
6.63E+05 1.45E-03 P1AE0408 2 + 1 head-to-tail 6.0 nM 2.91E+05
1.74E-03 format P1AE0637 4 + 1 with C-terminal 7.3 nM 2.82E+05
2.05E-03 crossFab Note: All K.sub.Ds are dependent from the
specific experimental conditions.
5.3.3 Binding to CD40 (Surface Plasmon Resonance)
[0823] The capacity of the bispecific constructs to bind human CD40
was assessed by surface plasmon resonance (SPR). All SPR
experiments were performed on a Biacore T200 (Biacore) at
25.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4,
0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, (Biacore).
[0824] In accordance with Example 4.2.7, association was measured
by injection of human CD40 extra cellular domain in various
concentrations in solution for 300 sec at a flow of 30 .mu.l/min
starting with 300 nM in 1:3 dilutions. The dissociation phase was
monitored for up to 1200 sec and triggered by switching from the
sample solution to running buffer. The surface was regenerated by
60 sec washing with a Glycine pH 2.1 solution at a flow rate of 30
.mu.l/min. Bulk refractive index differences were corrected by
subtracting the response obtained from a goat anti human
F(ab').sub.2 surface. Blank injections are also subtracted (=double
referencing). For calculation of apparent K.sub.D and other kinetic
parameters the Langmuir 1:1 model was used. The apparent Kd was
calculated using the Biacore.TM. B4000 evaluation software (version
1.1).
TABLE-US-00034 TABLE 31 Binding of exemplary bispecific CD40
.times. FAP antigen binding molecules to recombinant human CD40_ECD
(Biacore) Ligand format description KD ka (1/Ms) kd (1/s) P1AE0192
1 + 1 crossMab 3.7 nM 2.09E+06 7.77E-03 P1AE0408 2 + 1 head-to-tail
format 3.6 nM 2.34E+06 8.43E-03 P1AE0637 4 + 1 C-terminal crossFab
4.0 nM 1.79E+06 7.22E-03 fusion Note: All K.sub.Ds are dependent
from the specific experimental conditions.
5.3.4 Binding to Human CD40-Expressing Daudi Cells
[0825] The binding to cell surface CD40 was tested using Daudi
cells, a human B lymphoblast cell line with high expression levels
of human CD40 (ATCC CCL-213) as described in Example 1.4.2.
Exemplary EC.sub.50 values as measured for some of the bispecific
antigen binding molecules comprising humanized CD40 antibody
variants are shown in Table 8.
5.3.5 Functional Properties of Bispecific Antigen Binding Molecule
Comprising Humanized CD40 Antibody Variants
[0826] The functional properties of the bispecific antigen binding
molecules comprising humanized CD40 antibody variants were analyzed
in accordance to the experiments described in Example 2. Exemplary
data are provided in Tables 9, 10 or 11 as shown herein before.
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
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180340030A1).
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
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180340030A1).
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