U.S. patent application number 17/693292 was filed with the patent office on 2022-06-23 for methods, kits and apparatus for expanding a population of cells.
This patent application is currently assigned to Juno Therapeutics GmbH. The applicant listed for this patent is Juno Therapeutics GmbH. Invention is credited to Lothar Germeroth, Christian Stemberger.
Application Number | 20220195388 17/693292 |
Document ID | / |
Family ID | 1000006193601 |
Filed Date | 2022-06-23 |
United States Patent
Application |
20220195388 |
Kind Code |
A1 |
Germeroth; Lothar ; et
al. |
June 23, 2022 |
METHODS, KITS AND APPARATUS FOR EXPANDING A POPULATION OF CELLS
Abstract
The present invention relates to in vitro-methods of expanding a
population of cells such as lymphocytes, comprising contacting a
sample comprising a population of cells with a multimerization
reagent. The multimerization reagent has reversibly immobilized
thereon (bound thereto) a first agent that provides a primary
activation signal to the cells and optionally, a second agent that
provides a co-stimulatory signal. The invention also provides
multimerization reagents, kits, arrangements and an apparatus for
expanding cells.
Inventors: |
Germeroth; Lothar; (Munich,
DE) ; Stemberger; Christian; (Munich, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Juno Therapeutics GmbH |
Munich |
|
DE |
|
|
Assignee: |
Juno Therapeutics GmbH
Munich
DE
|
Family ID: |
1000006193601 |
Appl. No.: |
17/693292 |
Filed: |
March 11, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15304045 |
Oct 14, 2016 |
11274278 |
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PCT/EP2015/058339 |
Apr 16, 2015 |
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17693292 |
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61980506 |
Apr 16, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 47/12 20130101;
C12M 47/02 20130101; C12N 5/0636 20130101 |
International
Class: |
C12N 5/0783 20060101
C12N005/0783; C12M 1/00 20060101 C12M001/00 |
Claims
1. An in vitro method of stimulating a population of cells, the
method comprising incubating a sample comprising a population of
cells in the presence of a multimerization reagent that is
reversibly bound to a first agent, wherein the first agent (i)
comprises at least one binding partner C1 that is reversibly bound
to a binding site Z1 of the multimerization reagent and (ii) binds
to a receptor molecule on the surface of cells in the population to
stimulate a signal in the cells.
2. The method of claim 1, wherein the multimerization reagent is
reversibly bound to a second agent, wherein the second agent (i)
comprises at least one binding partner C2 that is reversibly bound
to a binding site Z2 of the multimerization reagent and (ii) binds
to an accessory molecule on the surface of the cells to stimulate
an accessory signal in the cells.
3. The method of claim 1, wherein the cells comprise T cells.
4. The method of claim 3, wherein the first agent stimulates a
TCR/CD3 complex-associated signal in the T cells.
5. The method of claim 3, wherein the first agent binds to CD3.
6. The method of claim 2, wherein the accessory molecule is CD28 or
CD137.
7. The method of claim 2, wherein the first agent binds to CD3, and
the second agent binds to CD28.
8. The method of claim 2, wherein: the first agent comprises an
anti-CD3 antibody, a divalent antibody fragment of an anti-CD3
antibody, a monovalent antibody fragment of an anti-CD3 antibody,
or a proteinaceous CD3 binding molecule with antibody-like binding
properties; and/or the second agent comprises an anti-CD28
antibody, a divalent antibody fragment of an anti-CD28 antibody, a
monovalent antibody fragment of an anti-CD28-antibody, a
proteinaceous CD28 binding molecule with antibody-like binding
properties, an anti-CD137 antibody, a divalent antibody fragment of
an anti-CD137 antibody, a monovalent antibody fragment of an
anti-CD137 antibody, a proteinaceous CD137 binding molecule with
antibody-like binding properties, 4-1BB ligand, or a mixture of any
of the foregoing.
9. The method of claim 2, wherein the first agent and/or the second
agent comprises a monovalent antibody fragment.
10. The method of claim 2, wherein the first agent comprises an
anti-CD3 antibody or antibody fragment, and the second agent
comprises an anti-CD28 antibody or antibody fragment.
11. The method of claim 2, wherein the first agent comprises an
anti-CD3 Fab, and the second agent comprises an anti-CD28 Fab.
12. The method of claim 1, wherein the multimerization reagent is
in soluble form.
13. The method of claim 1, wherein the multimerization reagent is
immobilized on a solid support.
14. The method of claim 1, wherein the multimerization reagent
comprises streptavidin, avidin, a mutein of streptavidin that
reversibly binds to the binding partner C1, a mutein of avidin that
reversibly binds to the binding partner C1, a reagent that
comprises at least two chelating groups K, wherein the at least two
chelating groups are capable of binding to a transition metal ion,
thereby rendering moiety A capable of binding to an oligohistidine
affinity tag, multimeric glutathione-S-transferase, multimeric
calmodulin or a biotinylated carrier protein.
15. The method of claim 1, wherein the multimerization reagent
comprises an oligomer or a polymer of streptavidin, an oligomer or
a polymer of avidin, an oligomer or a polymer of a mutein of
streptavidin that reversibly binds to the binding partner C1, or an
oligomer or a polymer of a mutein of avidin that reversibly binds
to the binding partner C1.
16. The method of claim 2, wherein the multimerization reagent
comprises an oligomer or a polymer of a mutein of streptavidin that
reversibly binds to the binding partners C1 and C2.
17. The method of claim 16, wherein the first agent comprises an
anti-CD3 antibody or antibody fragment, and the second agent
comprises an anti-CD28 antibody or antibody fragment.
18. The method of claim 16, wherein the streptavidin mutein
comprises the amino acid sequence
Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 or
Ile.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47 at sequence positions
44 to 47 of wild type streptavidin.
19. The method of claim 18, wherein the N-terminal amino acid
residue of the streptavidin mutein is in the region of sequence
positions 10 to 16 of the wildtype streptavidin amino acid
sequence, and the C-terminal amino acid residue of the streptavidin
mutein is in the region of sequence positions 133 to 142 of the
wildtype streptavidin amino acid sequence.
20. The method of claim 18, wherein the first agent comprises an
anti-CD3 antibody or antibody fragment, and the second agent
comprises an anti-CD28 antibody or antibody fragment.
21. The method of claim 1, wherein: (a) the binding partner C1
comprises biotin, and the multimerization reagent comprises a
streptavidin mutein that reversibly binds to biotin or an avidin
mutein that reversibly binds to biotin; (b) the binding partner C1
comprises a biotin analog that reversibly binds to streptavidin or
avidin, and the multimerization reagent comprises streptavidin,
avidin, a streptavidin mutein that reversibly binds to the biotin
analog, or an avidin mutein that reversibly binds to the biotin
analog; or (c) the binding partner C1 comprises a streptavidin or
avidin binding peptide, and the multimerization reagent comprises
streptavidin, avidin, a streptavidin mutein that reversibly binds
to the streptavidin or avidin binding peptide, or an avidin mutein
that reversibly binds to the streptavidin or avidin binding
peptide.
22. The method of claim 1, wherein the binding partner C1 comprises
a streptavidin-binding peptide, and the multimerization reagent
comprises a streptavidin mutein that reversibly binds to the
streptavidin-binding peptide.
23. The method of claim 22, wherein the streptavidin-binding
peptide is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1) or
SAWSHPQFEK(GGGS).sub.2GGSAWSHPQFEK (SEQ ID NO: 7).
24. The method of claim 2, wherein the binding partner C1 comprises
a first streptavidin-binding peptide, the binding partner C2
comprises a second streptavidin-binding peptide, and the
multimerization reagent comprises a streptavidin mutein that
reversibly binds to the first and second streptavidin-binding
peptides.
25. The method of claim 24, wherein the multimerization reagent
comprises an oligomer or a polymer of the mutein of
streptavidin.
26. The method of claim 24, wherein the first agent comprises an
anti-CD3 antibody or antibody fragment, and the second agent
comprises an anti-CD28 antibody or antibody fragment.
27. The method of claim 24, wherein the streptavidin mutein
comprises the amino acid sequence
Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 or
Ile.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47 at sequence positions
44 to 47 of wild type streptavidin.
28. The method of claim 27, wherein the N-terminal amino acid
residue of the streptavidin mutein is in the region of sequence
positions 10 to 16 of the wildtype streptavidin amino acid
sequence, and the C-terminal amino acid residue of the streptavidin
mutein is in the region of sequence positions 133 to 142 of the
wildtype streptavidin amino acid sequence.
29. The method of claim 27, wherein the multimerization reagent
comprises an oligomer or a polymer of the mutein of
streptavidin.
30. The method of claim 27, wherein the first agent comprises an
anti-CD3 antibody or antibody fragment, and the second agent
comprises an anti-CD28 antibody or antibody fragment.
31. The method of claim 2, wherein: the binding partner C1
comprises a streptavidin-binding peptide that is
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1) or
SAWSHPQFEK(GGGS).sub.2GGSAWSHPQFEK (SEQ ID NO: 7), the binding
partner C2 comprises a streptavidin-binding peptide that is
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1) or
SAWSHPQFEK(GGGS).sub.2GGSAWSHPQFEK (SEQ ID NO: 7), and the
multimerization reagent comprises a streptavidin mutein comprising
the amino acid sequence Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47
or Ile.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47 at sequence
positions 44 to 47 of wild type streptavidin.
32. The method of claim 1, further comprising disrupting the
binding between the binding partner C1 of the first agent and the
binding site Z1 of the multimerization reagent.
33. The method of claim 32, wherein the reversible bond is
disrupted by contacting the cells with biotin or a biotin analog,
wherein: (a) the binding partner C1 comprises biotin, and the
multimerization reagent comprises a streptavidin mutein that
reversibly binds to biotin or an avidin mutein that reversibly
binds to biotin, (b) the binding partner C1 comprises a biotin
analog that reversibly binds to streptavidin or avidin, and the
multimerization reagent comprises streptavidin, avidin, a
streptavidin mutein that reversibly binds to the biotin analog, or
an avidin mutein that reversibly binds to the biotin analog, or (c)
the binding partner C1 comprises a streptavidin or avidin binding
peptide, and the multimerization reagent comprises streptavidin,
avidin, a streptavidin mutein that reversibly binds to the
streptavidin or avidin binding peptide, or an avidin mutein that
reversibly binds to the streptavidin or avidin binding peptide.
34. The method of claim 16, further comprising disrupting the
binding between the streptavidin mutein and the binding partners C1
and C2, wherein the disrupting is by contacting the cells with
biotin.
35. The method of claim 24, further comprising disrupting the
binding between the streptavidin mutein and the first and second
streptavidin-binding peptides, wherein the disrupting is by
contacting the cells with biotin.
36. The method of claim 3, wherein a T cell receptor or a chimeric
antigen receptor is introduced into the T cells either after the
stimulation or during the stimulation.
37. A reagent kit comprising: (i) a multimerization reagent,
wherein the multimerization reagent comprises at least one binding
site Z1 for the reversible binding of a first agent; and (ii) a
first agent capable of binding to a receptor molecule on the
surface of cells and comprising at least one binding partner C1
able to reversibly bind to the binding site Z1 of the
multimerization reagent, wherein binding of the first agent to the
receptor molecule on the surface of the cells stimulates a signal
to the cells.
38. An arrangement of a bioreactor and a stationary phase for
chromatography, wherein the bioreactor is suitable for the
expansion of cells, wherein the stationary phase is suitable for
cell separation and removal of reagents, the stationary phase being
a gel filtration matrix and/or affinity chromatography matrix,
wherein the gel filtration and/or affinity chromatography matrix
comprises an affinity reagent, the affinity reagent comprising (i)
a binding site Z1 capable of specifically binding to a binding
partner C1 and/or (ii) a binding site Z2 capable of specifically
binding to a binding partner C2, the affinity reagent thereby being
suitable for immobilizing the first binding partner C1 and/or the
second binding partner C2 on the stationary phase, and wherein the
bioreactor and the stationary phase are fluidly connected.
39. An apparatus for purification and expansion of target cells,
the apparatus comprising the arrangement of claim 38.
40. A multimerization reagent, wherein the multimerization reagent
is in soluble form and is reversibly bound to a first agent,
wherein the first reagent (i) comprises at least one binding
partner C1 that is reversibly bound to a binding site Z1 of the
multimerization reagent, and (ii) is capable of binding to a
receptor molecule on the surface of cells to stimulate a signal in
the cells.
41. A composition comprising: (1) a first multimerization reagent,
wherein the first multimerization reagent is in soluble form and
comprises at least one binding site Z1 for the reversible binding
of a first agent, the first agent (i) comprising at least one
binding partner C1 that is reversibly bound to at least one binding
site Z1 of the multimerization reagent and (ii) being capable of
binding to a receptor molecule on the surface of cells to stimulate
a signal in the cells; and (2) a second multimerization reagent,
wherein the second multimerization reagent is in soluble form and
comprises at least one binding site Z2 for the reversible binding
of a second agent, the second agent (i) comprising at least one
binding partner C2 that is reversibly bound to at least one binding
site Z2 of the multimerization reagent and (ii) being capable of
binding to an accessory molecule on the surface of the cells to
stimulate an accessory signal in the cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/304,045 filed 14 Oct. 2016, which is a U.S.
National Stage of International Application No. PCT/EP2015/058339
filed 16 Apr. 2015, which claims the benefit of priority to U.S.
Provisional Application No. 61/980,506 filed 16 Apr. 2014, entitled
"Methods, Kits And Apparatus For Expanding A Population Of Cells",
the contents of each of which are hereby incorporated by reference
in their entirety for all purposes.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The present application is being filed with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled 735042004001SeqList.txt, created on Mar. 7, 2022,
which is 24,576 bytes in size. The information in electronic format
of the Sequence Listing is incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to the expansion
(proliferation) of a population of cells such as a population of
lymphocytes. The invention in general provides novel methods and
reagents for the expansion (proliferation) of cell populations that
require binding of a receptor binding molecule (such as a first
agent as described herein) to a receptor molecule on the surface of
the cells, thereby providing a primary activation signal to the
cells. The invention employs a multimerization reagent that has
immobilized thereon (bound thereto) a first agent that provides a
primary activation signal to the cells. This primary activation
signal may as such be sufficient to activate the cells to
expand/proliferate. This first agent can either be bound reversibly
or also irreversibly to the multimerization reagent. The
multimerization reagent may have immobilized thereon (bound
thereto) also a second agent that stimulates an accessory molecule
on the surface of the cells. The second agent, when binding to the
accessory molecule on the surface on the surface of the cells, may
thereby stimulate the activated cells to expand. Also this second
agent can either be bound reversibly or also irreversibly to the
multimerization reagent. The multimerization agent may either be
immobilized on a solid support or soluble. In one aspect, the
method disclosed herein is a serial expansion of a population of
cells in which a complete population of lymphocytes is
stimulated/expanded, the reagents necessary for the expansion are
then removed by chromatography on a suitable stationary phase and
the expanded/stimulated cells are optionally transfected with e.g.
a T cell receptor or a chimeric antigen receptor (CAR) and
subjected to a second stimulation expansion with a different
stimulatory molecule that binds to the introduced T cell receptor
or the chimeric antigen receptor. The invention also relates to an
apparatus for the expansion of the selected cell population.
BACKGROUND OF THE INVENTION
[0004] The development of techniques for propagating T cell
populations in vitro has been crucial to many of the advances in
the understanding of T cell recognition of antigen and T cell
activation. The development of culture methods for the generation
of human antigen-specific T cell clones has been useful in defining
antigens expressed by pathogens and tumors that are recognized by T
cells to establish methods of immunotherapy to treat a variety of
human diseases. Antigen-specific T cells can be expanded in vitro
for use in adoptive cellular immunotherapy or cancer therapy in
which infusions of such T cells have been shown to have anti-tumor
reactivity in a tumor-bearing host. In addition, adoptive
immunotherapy has also been used to treat viral infections in
immunocompromised individuals.
[0005] A method of expanding human T cells in vitro in the absence
of exogenous growth factor and accessory cells that has been
established in the recent years is described in U.S. Pat. No.
6,352,694 B1 and European Patent EP 0 700 430 B1. Disclosed in
these patents is an in vitro method for inducing a population of T
cells to proliferate. The method comprises contacting a population
of T cells with a solid phase surface having directly immobilized
thereon: (a) a first agent which provides a primary activation
signal to the T cells, thereby activating the T cells; and (b) a
second agent which stimulates an accessory molecule on the surface
of the T cells, thereby stimulating the activated T cells. The
binding of the first agent and the second agent to the T cells
induces the T cells to proliferate/to expand. The preferred first
agent described in U.S. Pat. No. 6,352,694 B1 and European Patent
EP 0 700 430 B1 is a monoclonal anti-CD3 antibody which binds to
the TCR/CD3 (TCR=T Cell Receptor) complex and thereby stimulates
the TCR/CD3 complex-associated signal in the T cells. The preferred
second agent according to these two patents is a monoclonal
anti-CD28 antibody which binds the accessory molecule CD28 that is
present on T cells. Binding of this second agent to the CD28
accessory molecule provides the necessary co-stimulus that is
necessary for expansion/proliferation of activated T cells.
Meanwhile, Dynabeads.RTM. CD3/CD28 (Invitrogen) are commercially
available for T cell expansion. Dynabeads.RTM. CD3/CD28 CTS.TM. are
uniform, 4.5 .mu.m superparamagnetic, sterile, non-pyrogenic
polystyrene beads coated with a mixture of affinity purified
monoclonal antibodies against the CD3 and CD28 cell surface
molecules on human T cells.
[0006] However, such magnetic beads are, for example, difficult to
integrate into a method to expand cells under conditions required
for clinical trials or therapeutic purposes since it has to be made
sure that these magnetic beads are completely removed before
administering the expanded T cells to a patient. Thus, the present
invention aims to provide an alternative method for expanding cell
populations such as regulatory T cells or central memory T-cells
for research, diagnostic and especially therapeutic purposes.
Ideally, this new method should also be compatible with integration
into an automatized process which can be used for rapid and easy
expansion of the desired cells population for therapeutic
applications.
[0007] This object is solved by the subject matter of the
independent claims, inter alia the methods, kits, arrangements and
apparatuses as recited in the independent claims.
SUMMARY OF THE INVENTION
[0008] The present invention provides methods, kits, arrangements,
and apparatus for the in vitro expansion of a desired cell
population, having a receptor molecule on its surface which can
provide upon binding of a suitable agent a primary activation
signal to the population of cells and thereby activating the
population of cells for expansion (proliferation). Thus, the
methods of the invention are also used for inducing a population of
cells to proliferate.
[0009] According to a first aspect, the invention provides an in
vitro-method of expanding a population of cells, comprising
contacting a sample comprising the population of cells with a
multimerization reagent, [0010] wherein the multimerization reagent
has reversibly immobilized thereon (bound thereto) a first agent
that provides a primary activation signal to the cells; [0011]
wherein the multimerisation reagent comprises at least one binding
site Z1 for the reversible binding of the first agent, [0012]
wherein the first agent comprises at least one binding partner C1,
wherein the binding partner C1 is able of reversibly binding to the
binding site Z1 of the multimerization reagent, wherein the first
agent is bound to the multimerization reagent via the reversible
bond formed between the binding partner C1 and the binding site Z1,
and wherein the first agent binds to a receptor molecule on the
surface of the cells, thereby providing a primary activation signal
to the cells and thereby activating the cells.
[0013] According to a second aspect the invention provides an in
vitro-method of expanding a population of cells, comprising
contacting a sample comprising the population of cells with a
multimerization reagent, [0014] wherein the multimerization reagent
is in a soluble form and has immobilized thereon (bound thereto) a
first agent that provides a primary activation signal to the cells;
[0015] wherein the multimerisation reagent comprises at least one
binding site Z1 for the binding of the first agent, [0016] wherein
the first agent comprises at least one binding partner C1, wherein
the binding partner C1 is able of binding to the binding site Z1 of
the multimerization reagent, wherein the first agent is bound to
the multimerization reagent via the bond formed between the binding
partner C1 and the binding site Z1, and [0017] wherein the first
agent binds to a receptor molecule on the surface of the cells,
thereby providing a primary activation signal to the cells and
thereby activating the cells.
[0018] According to a third aspect the invention provides a reagent
kit for expanding a population of cells, the kit comprising [0019]
(i) a multimerization reagent, wherein the multimerisation reagent
comprises at least one binding site Z for the reversible binding of
a first agent, [0020] (ii) a first agent that binds to a receptor
molecule on the surface of the cells, thereby providing a primary
activation signal to the cells and thereby activating the cells,
[0021] wherein the first agent comprises at least one binding
partner C1, wherein the binding partner C1 is able of reversibly
binding to a binding site Z1 of the multimerization reagent,
wherein the first agent is bound to the multimerization reagent via
the reversible bond formed between the binding partner C1 and the
binding site Z1, and [0022] (iii) a second agent that stimulates an
accessory molecule on the surface of the cells, [0023] wherein the
second agent comprises a binding partner C2, wherein the binding
partner C2 is able of reversibly binding to a binding site Z2 of
the multimerization reagent, wherein the second agent is bound to
the multimerization reagent via the bond formed between the binding
partner C2 and the binding site Z2, [0024] wherein the second agent
binds to the accessory molecule on the surface on the surface of
the cells, thereby stimulating the activated cells.
[0025] According to a fourth aspect the invention provides a
reagent kit for expanding a population of cells, the kit comprising
[0026] (i) a multimerization reagent, [0027] wherein the
multimerisation reagent is in soluble form and comprises at least
one binding site Z for the reversible binding of a first agent,
[0028] (ii) a first agent that binds to a receptor molecule on the
surface of the cells, thereby providing a primary activation signal
to the cells and thereby activating the cells, [0029] wherein the
first agent comprises at least one binding partner C1, wherein the
binding partner C1 is able of binding to a binding site Z1 of the
multimerization reagent, wherein the first agent is bound to the
multimerization reagent via the reversible bond formed between the
binding partner C1 and the binding site Z1.
[0030] According to a fifth aspect the invention provides an in
vitro-method of serially expanding a population of lymphocytes,
wherein the population of lymphocytes comprises T cells, the method
comprising [0031] contacting a sample comprising the T cell
comprising population of lymphocytes with a multimerization
reagent, [0032] wherein the multimerization reagent is in a soluble
form and has reversibly immobilized thereon (i) a first agent that
provides a primary activation signal to the T cells and (ii) a
second agent which stimulates an accessory molecule on the surface
of the T cells, [0033] wherein the multimerisation reagent
comprises at least one binding site Z1 for the reversible binding
of the first agent, [0034] wherein the first agent comprises at
least one binding partner C1, wherein the binding partner C1 is
able of reversibly binding to the binding site Z1 of the
multimerization reagent, wherein the first agent is bound to the
multimerization reagent via the reversible bond formed between the
binding partner C1 and the binding site Z1, [0035] wherein the
multimerisation reagent comprises at least one binding site Z2 for
the reversible binding of the second agent, [0036] wherein the
second agent comprises at least one binding partner C2, wherein the
binding partner C2 is able of reversibly binding to the binding
site Z2 of the multimerization reagent, wherein the first agent is
bound to the multimerization reagent via the reversible bond formed
between the binding partner C2 and the binding site Z2, [0037]
wherein the first agent binds to a receptor molecule on the surface
of the T cells, thereby providing a primary activation signal to
the cells and thereby activating the T cells, [0038] wherein the
second agent binds to the accessory molecule on the surface of the
T cells, thereby stimulating the activated cells, the first agent
and the second agent thereby together inducing the T cells to
expand.
[0039] According to a sixth aspect the invention provides an
arrangement of a bioreactor and a stationary phase for
chromatography, [0040] wherein the bioreactor is suitable for the
expansion of cells, [0041] wherein the stationary phase is suitable
for cell separation and removal of reagents, the stationary phase
being a gel filtration matrix and/or affinity chromatography
matrix, wherein the gel filtration and/or affinity chromatography
matrix comprises an affinity reagent, wherein the affinity reagent
comprises a binding site Z1 specifically binding to a binding
partner C1 comprised in a first agent and/or the affinity reagent
comprises a binding site Z2 specifically binding to a binding
partner C2 comprised in a second agent, thereby being suitable of
immobilizing on the stationary phase the first agent and/or the
second agent, the first binding partner C1 and/or the free second
binding partner C2, [0042] wherein the bioreactor and the
stationary phase are fluidly connected.
[0043] According to a seventh aspect the invention provides an
apparatus for purification and expansion of a population of cells,
the apparatus comprising at least one arrangement of a bioreactor
and a stationary phase for chromatography according to the sixth
aspect.
[0044] According to an eight aspect, the invention provides a
multimerization reagent capable of expanding a population of cells,
[0045] wherein the multimerisation reagent is in soluble form and
comprises at least one binding site Z1 for the reversible binding
of a first agent that provides a primary activation signal to the
cells, [0046] wherein the multimerization reagent has reversibly
immobilized thereon (bound thereto) said first agent that provides
a primary activation signal to the cells;
[0047] wherein the first agent comprises at least one binding
partner C1, wherein the binding partner C1 is able of reversibly
binding to the at least one binding site Z1 of the multimerization
reagent, wherein the first agent is bound to the multimerization
reagent via the reversible bond formed between the binding partner
C1 and the binding site Z1,
[0048] According to a ninth aspect, the invention provides a
composition capable of expanding a population of cells, the
composition comprising [0049] (i) a first multimerization reagent,
[0050] wherein the first multimerisation reagent is in soluble form
and comprises at least one binding site Z1 for the reversible
binding of a first agent that provides a primary activation signal
to the cells, [0051] wherein the first multimerization reagent has
reversibly immobilized thereon (bound thereto) said first agent
that provides a primary activation signal to the cells; [0052]
wherein the first agent comprises at least one binding partner C1,
wherein the binding partner C1 is able of reversibly binding to the
at least one binding site Z1 of the multimerization reagent,
wherein the first agent is bound to the multimerization reagent via
the reversible bond formed between the binding partner C1 and the
binding site Z1, and [0053] (ii) a second multimerization reagent,
[0054] wherein the second multimerization reagent is in soluble
form and comprises at least one binding site Z2 for the reversible
binding of a second agent that stimulates an accessory molecule on
the surface of the cells, [0055] wherein the multimerization
reagent has reversibly immobilized thereon (bound thereto) said
second agent that stimulates an accessory molecule on the surface
of the cells, [0056] wherein the second agent comprises a binding
partner C2, wherein the binding partner C2 is able of binding to
the at least one binding site Z2 of the multimerization reagent,
wherein the second agent is bound to the multimerization reagent
via the bond formed between the binding partner C2 and the binding
site Z2.
DESCRIPTION OF THE DRAWINGS
[0057] The invention will be better understood with reference to
the detailed description when considered in conjunction with the
non-limiting examples and the accompanying drawings. The figures
illustrate embodiments of methods of the invention. Without wishing
to be bound by theory, the figures include conclusions with regard
to the underlying expansion mechanism. The conclusions are given
for illustrative purposes only and merely serve in allowing a
visualization of the expansion method is achievable on a molecular
level.
[0058] FIG. 1 depicts an embodiment of an in vitro-method of
expanding of expanding a population of cells that has a cell
surface receptor the binding of which by a first agent can provide
an activation signal for the cells to expand.
[0059] As shown in FIG. 1a a sample that comprises the population
of cells (2) that carry a surface receptor molecule (30) is
contacted with a multimerization reagent (4). The population of
cells (2) is in mixture with other cell populations (22) that lack
the surface receptor molecule (30). The multimerization reagent (4)
has reversibly immobilized thereon (bound thereto) a first agent
(6) that provides a primary activation signal to the cells. The
multimerization reagent (4) comprises at least one binding site Z1
(42) for the reversible binding of the first agent (6) and the
first agent (6) comprises at least one binding partner C1 (6a),
wherein the binding partner C1 (6a) is able of reversibly binding
to the binding site Z1 (44) of the multimerization reagent. Thus,
for immobilization, the first agent (6) is bound to the
multimerization reagent (4) via the reversible bond formed between
the binding partner C1 (6a) and the binding site Z1 (42). In the
example shown in FIG. 1 the multimerization reagent (4) has a
second binding site Z2 (44) which is not used in this example. The
multimerization reagent (4) is itself immobilized on a solid
support (10) such as a magnetic bead, a polymeric bead of a surface
of a cell culture plate or reactor. The population of cells (2)
can, for example be, a lymphocyte cell population such as a
population of B cells that can be activated via the CD40 receptor
(see, for example, Carpenter et al, Journal of Translational
Medicine 2009, 7:93 "Activation of human B cells by the agonist
CD40 antibody CP-870,893 and augmentation with simultaneous
toll-like receptor 9 stimulation). In this case, the cell surface
molecule (30) is CD40 and the first reagent (6) can be any CD40
binding molecule that provides the desired activation signal, for
example, the monoclonal antibody CP-870,893 or an antibody binding
fragment thereof such an a monovalent Fab fragment. The binding
partner C1 of the first agent (6) may, for example, be any affinity
peptide that is fused or conjugated to, for example, the C-terminus
of one the two polypeptide chains (heavy or light chain) of the
antibody molecule. The binding partner C1 (6a) may, for example, be
a streptavidin-binding peptide such as the peptide
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 01), also known as the
"Strep-tag.RTM.") that is described in U.S. Pat. No. 5,506,121, for
example, or streptavidin binding peptides having a sequential
arrangement of two or more individual binding modules as described
in International Patent Publication WO 02/077018 or U.S. Pat. No.
7,981,632. When using a streptavidin binding peptide as binding
partner C1, the multimerization reagent (4) be any streptavidin
mutein to which the streptavidin peptide (=first binding partner C1
(6a)) reversibly binds via its (biotin) binding sites Z1 (42)
schematically shown in FIG. 1. Such a multimerization reagent may
be a streptavidin mutein (analog) that comprises the amino acid
sequence Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 (SEQ ID NO:
02) at sequence positions 44 to 47 of wild type streptavidin or a
streptavidin mutein (analog) that comprises the amino acid sequence
lle.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47 (SEQ ID NO: 03) at
sequence positions 44 to 47 of wild type streptavidin, both of
which are described in U.S. Pat. No. 6,103,493, for example, and
are commercially available under the trademark Strep-Tactin.RTM..
In the Example of FIG. 1, the multimerization reagent (4) might
further include multimeric calmodulin or glutathione-S-transferase,
both of which form reversible bonds with calmodulin binding
peptides or glutathione. Thus, the binding site Z2 (44) can be
formed by calmodulin or glutathione-S-transferase. Such a protein
conjugate of for example, calmodulin with a streptavidin mutein can
be made by standard protein chemistry, for example, by using
bifunctional linkers.
[0060] As shown in FIG. 1b, after contacting the cell population
(2) with the multimerisation reagent (4) and usually incubating the
cell population with the multimerization reagent (4), the
population of cells (2) forms complexes/is bound to the
multimerization agent via the first agent (6). The first agent
binds specifically to the cell surface receptor molecule such as
CD40 in this Example and provides the activation signal for cell
expansion, of for example B cells. The other cell populations (22)
contained in the initial sample that lack the specific cell surface
molecule (30) do not bind to the multimerization reagent. In this
respect, it is noted that the cell population (2) usually has
multiple copies of the cell surface molecule (30) on its surface
and binding of these multiple copies is typically needed for
activation. Thus, the multimerization agent (4) provide typically
more than one binding site Z1 so that multiple first agents (6) can
be reversibly bound to achieve "multimerization" of the first
agent, meaning to present the first agent in a sufficient density
to the population of cells (2) (not shown in the scheme of FIG. 1).
In this respect, it is noted that a multimerization agent as used
herein can as such have multiple binding sites Z1, for example, a
streptavidin mutein (being a homo-tetramer) in its native state has
four such binding sites Z1. It is however also possible that the
multimerization reagent is based on a compound that has as such
only one binding site Z1 for the reversible binding of a binding
partner C1. Such an example is multimeric calmodulin. Calmodulin as
such has only one binding site for calmodulin binding peptides.
However, calmodulin can be biotinylated and then reacted with
streptavidin-oligomers (see also below), thereby providing a
multimerization reagent in which multiple calmodulin molecules are
presented in high density on a "scaffold", thereby providing
multimeric calmodulin.
[0061] As shown in FIG. 1c, after incubation (which is usually
carried out over a period of time suitable to achieve expansion of
the desired cell population) the binding between the binding
partner C1 (6a) of the first agent (6) and the binding site Z1 of
the multimerization reagent (4) is disrupted by disrupting the
respective reversible bond. The disruption may be achieved by
adding a competitor to the incubation/reaction mixture containing
the population of cells (2) being bound to the multimerization
reagent. For competitive disruption (which can be understood as
being a competitive elution) of the reversible bond between the
binding partner C1 (6a) of the first agent and the binding site Z1
(22) of the multimerization reagent, the incubation
mixture/population of cells can be contacted with a free first
binding partner C1 (20) or an analog of said first binding partner
C that is capable of disrupting the bond between the first binding
partner C1 (6a) and the binding site Z1 (22). In the example of the
binding partner C1 being a streptavidin binding peptide that binds
to biotin binding site of streptavidin, the first free partner C1
(20) may be the corresponding free streptavidin binding peptide or
an analogue that binds competitively. Such an analogue can, for
example, be biotin or a biotin derivate such as desthiobiotin.
[0062] As shown in FIG. 1d, addition of the first free partner (20)
or the analogue thereof results in displacement of the binding
partner C1 (6a) from the multimerization reagent (4) and thus,
since the binding partner C1 is comprised in the first agent (6),
displacement of the first agent (6) from the multimerization
reagent (4). This displacement of the first agent (6) in turn
results in a dissociation of the first agent (6) from the cell
surface receptor (30), in particular if the binding affinity of the
bond between the first agent and the cell surface receptor (30) has
a dissociation constant (Ka) in the range of 10.sup.-2 M to
10.sup.-13 M and is thus also reversible. Due to this dissociation,
the stimulation of the cell population (2) is also terminated.
Thus, the present invention provides the advantage that the time
period of the stimulation or expansion of the cell population can
be exactly controlled and thus also the functional status of the
cell population can be closely controlled. In this context, it is
noted that the binding affinity of antibody molecules towards their
antigen, including for example, a cell surface receptor molecule
such as CD40 in this Example, is usually in the affinity range of
the K.sub.d of 10.sup.-7 M to 10.sup.-13 M. Thus, conventional
monoclonal antibodies can be used as first agent (and also of
course second agent as explained below) in the present invention.
In order to avoid any unwanted avidity effects that lead to a
stronger binding, monoclonal antibodies can also be used in form of
their monovalent antibody fragments such as Fab-fragments or single
chain Fv fragments.
[0063] In addition, due to the dissociation of the first agent from
the cell surface molecule (30), the present invention has the added
advantage that the stimulated cell population is free of
stimulating agents at the end of the stimulation period and that
all other reagents used in the method, namely the first agent (6)
as well as the free first partner (20) of the binding partner C1 or
the analogue thereof can be easily removed from the stimulated cell
population (2) via a "removal cartridge" described in International
patent application WO 2013/124474 while the multimerization reagent
(4) being immobilized on a solid support such as a bioreactor
surface or a magnetic bead is being held back. Thus, reverting to
the removal of the free agent (6) and the free first partner (20),
in accordance with the description of the "removal cartridge" in WO
2013/124474 (see with reference to FIG. 4 thereof, for example),
the elution sample obtained in
[0064] FIG. 1d here can be loaded onto the second chromatography
column of WO 2013/124474. This chromatography column has a suitable
stationary phase that is both an affinity chromatography matrix
and, at the same time, can act as gel permeation matrix. This
affinity chromatography matrix has an affinity reagent immobilized
thereon. The affinity reagent may, in the case of the current
Example, for instance, be streptavidin, a streptavidin mutein,
avidin, an avidin mutein or a mixture thereof. The first agent (6),
the free first partner (20) of the binding partner C1 (which is
also called "competition reagent" herein) bind to the affinity
reagent, thereby being immobilized on the chromatography matrix. As
a result the elution sample containing the isolated and expanded
cell population (2) is being depleted of the first agent (6) and
the competition reagent (20). The expanded cell population (2),
being freed of any reactants, is now in a condition for further
use, for example, for diagnostic applications (for example, further
FACS.TM. sorting) or for any cell based therapeutic
application.
[0065] FIG. 2 shows a further embodiment of an expansion method of
the invention. As shown in FIG. 2a a sample comprises a population
of cells (2) that carry two specific cell surface molecules (30)
and (32). The cell surface molecule (30) is involved in a primary
activation signal to the cell population, while the cell surface
molecule (32) is an accessory molecule on the cell surface that is
involved in providing a stimulus to the cells. The population of
cells may, for example, be a T cell population in which the cell
surface molecule (30) is a TCR/CD3 complex and the cell surface
molecule (32) is the accessory molecule CD28. Binding of both the
TCR/CD3 complex as the primary activation signal and CD28 as
co-stimulant are necessary for expansion/proliferation of T cells.
The population of T cells (2) is in mixture with other cell
populations (22) that lack the surface receptor molecules (30) and
(32). Also in this embodiment, the cell population (2) is contacted
with a multimerization reagent (4). The multimerization reagent (4)
has reversibly immobilized thereon (bound thereto) a first agent
(6) that provides a primary activation signal to the cells. In
addition, the multimerization agent has reversibly immobilized
thereon (bound thereto) a second agent (8) that stimulates CD28 as
accessory molecule on the surface of the cells.
[0066] The multimerization reagent (4) comprises at least one
binding site Z1 (42) for the reversible binding of the first agent
(6) and the first agent (6) comprises at least one binding partner
C1 (6a), wherein the binding partner C1 (6a) is able of reversibly
binding to the binding site Z1 (44) of the multimerization reagent.
Thus, for immobilization, the first agent (6) is bound to the
multimerization reagent (4) via the reversible bond formed between
the binding partner C1 (6a) and the binding site Z1 (42). In
addition, in the Example illustrated in FIG. 2, the second agent
(8) comprises a binding partner C2 (8a), wherein the binding
partner C2 is able of being reversibly bound to a binding site Z2
(44) of the multimerization reagent (4). The second agent (8) is
bound to the multimerization reagent (4) via the reversible bond
formed between the binding partner C2 (8a) and the binding site Z2
(44). In this Example, the first agent (6) might be a monoclonal
anti-CD3-antibody or an antigen binding fragment thereof such as a
Fab fragment. The second agent (8) might be a monoclonal anti-CD28
antibody or an antigen binding fragment thereof such as Fab
fragment. The first binding partner (6a) might be a streptavidin
binding peptide (6a) that is fused or conjugated to the anti-CD3
antibody or the anti-CD3 antibody fragment. The second binding
partner (8a) might be calmodulin binding peptide that is also
conjugated or fused to the CD28 antibody or the CD28 binding
antibody fragment. In this context, it is noted that monoclonal
antibodies against, for example, CD3 or CD28 are well-known (see,
for example, U.S. Pat. No. 6,352,694 B or European Patent EP 0 700
430 B1 discussed above) and are commercially available from
numerous suppliers such as Santa Cruz Biotechnology (Santa Cruz,
Calif., USA), Life Technologies, (Carlsbad, Calif., USA), BD
Biosciences (San Jose, Calif., USA), Biolegend (San Diego, Calif.,
USA) or Miltenyi Biotec (Bergisch Gladbach, Germany) to name only a
few. Accordingly, such monoclonal antibodies can be used as first
and second agent and can, for example, be chemically coupled
(conjugated) with a binding partner C1 or C2. Alternatively, it is
also possible to either clone the genes of the variable domains
from the hydridoma cell line or use an antibody of which the amino
acid sequence is known and produce a respective antibody fragment
such as a Fab fragment or a Fv recombinantly. When using such an
approach as described herein in the Example section for both the
hybridoma cell line OKT3 (ATCC.RTM. CRL-8001.TM., described in U.S.
Pat. No. 4,361,549) that produces a monoclonal anti-CD3 antibody)
and the anti-CD28 antibody 28.3 described by Vanhove et al, BLOOD,
15 Jul. 2003, Vol. 102, No. 2, pages 564-570 and GenBank accession
number AF451974.1, the binding partners C1 and C2 are conveniently
provided by the respective expression vector used for the
recombinant production so that the antibody fragment carries the
binding partner C1 or C2 as a fusion peptide as the C-terminus of
either the light or the heavy chain (In this context, the amino
acid sequence of the variable domain of the heavy chain and of the
variable domain of the light chain of the antibody OKT3 that are
described in Arakawa et al J. Biochem. 120, 657-662 (1996) are
shown for illustration purposes as SEQ ID NOS 17 and 18 and in the
accompanying Sequence Listings, while the amino acid sequence of
the variable domain of the anti-CD28 antibody 28.3 described by
Vanhove et al, supra, is shown as SEQ ID NOS 19 (VH) and 20 (VL) in
the accompanying Sequence Listings). Also this methodology of
cloning the variable domains of an antibody molecule and
recombinantly producing a respective antibody fragment is well
known to the person skilled in the art, see for example, Skerra, A.
(1994) A general vector, pASK84, for cloning, bacterial production,
and single-step purification of antibody Fab fragments. Gene 141,
79-84, or Skerra, A. (1993) Bacterial expression of immunoglobulin
fragments. Curr Opin Immunol. 5, 256-562). Finally, it is also
possible to generate antibody molecules of artificial binding
molecules with antibody like properties against a given target such
as CD3 or CD28 as in the Example of FIG. 2 by well-known evolutive
methods such as phage display (reviewed, e.g., in Kay, B. K. et al.
(1996) Phage Display of Peptides and Proteins--A Laboratory Manual,
1st Ed., Academic Press, New York N.Y.; Lowman, H. B. (1997) Annu.
Rev. Biophys. Biomol. Struct. 26, 401-424, or Rodi, D. J., and
Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93), ribosome
display (reviewed in Amstutz, P. et al. (2001) Curr. Opin.
Biotechnol. 12, 400-405) or mRNA display as reported in Wilson, D.
S. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 3750-3755.
[0067] In the case of the Example shown in FIG. 2, the
multimerization reagent (4) has two different binding sites Z1 (42)
and Z2 (44). With the binding partner C1 (6a) being a streptavidin
binding peptide, the binding site Z1 (42) of the multimerization
reagent (4) is provided by a suitable streptavidin mutein to which
the streptavidin peptide (6a) reversibly binds. Since the binding
C2 is a calmodulin binding peptide, the binding site Z2 (44) of the
multimerization reagent (4) is provided by multimeric calmodulin.
The multimerization reagent (4) can be a single molecule, for
example a conjugate of multimeric calmodulin with streptavidin
(this alternative would be usually used in case of a soluble
multimerization) or can also consist of two independent molecules.
The latter option is preferred when the multimerization reagent
(44) is immobilized on a solid support as shown in FIG. 2 In this
case, a mixture of a streptavidin mutein and calmodulin can be
coated (immobilized) on the solid support, for example, in a 1:1
molar ratio with respect to the binding sites Z1 and Z2. In this
context, it is noted that, due to the immobilisation of calmodulin
on the surface of the solid support, there is no need to prepare
multimeric calmodulin as explained above but immobilization of the
calmodulin on the surface is sufficient to present calmodulin
(that, as mentioned above, has only a single binding site for
calmodulin binding peptides, in a sufficiently high density to
ensure binding of the cell population (2). For example, in this
case, a bivalent antibody fragment that has two binding sites
against CD28 or an intact antibody that has per se two identical
binding sites could be used as second reagent (8).
[0068] As shown in FIG. 2b, after contacting the T cell population
(2) with the multimerisation reagent (4) and usually incubating the
cell population with the multimerization reagent (4), the
population of T cells (2) forms complexes/is bound to the
multimerization agent via the first agent (6) and the second agent
(8). The first agent (6) and the second agent (8) bind specifically
to the TCR/CD3 complex and the accessory molecule CD28, thereby
inducing the T cells to proliferate/expand.
[0069] As shown in FIG. 2c, after incubation (which is usually
carried out over a period of time suitable to achieve expansion of
the desired cell population) the binding between the binding
partner C1 (6a) of the first agent (6) and the binding site Z1 of
the multimerization reagent (4) is disrupted by disrupting the
respective reversible bond. Likewise, the binding between the
binding partner C2 (8a) of the second agent (8) and the binding
site Z2 of the multimerization reagent (4) is disrupted by
disrupting the respective reversible bond. The reversible bond
between the binding partner C1 (6a) of the first agent (6) and the
binding site Z1 can be disrupted by biotin (which acts as an
analogue (20) of the free first partner) while the reversible bond
between the binding partner C2 (8a) of the first agent (8) and the
binding site Z2 can be disrupted by the addition of a metal
chelator (calcium chelator) such as EDTA or EGTA (that acts an
analogue (20) of the free second partner) since the binding of
calmodulin to calmodulin binding peptides is calcium ion
(Ca.sup.2+) dependent). This of course means that the contacting of
the cell population (2) is carried out in a Ca.sup.2+ containing
buffer.
[0070] As shown in FIG. 2d, addition of the analogue (20) of the
first free partner and the second free partner, respectively
results in displacement of the binding partners C1 (6a) and C2 (8a)
from the multimerization reagent (4) and thus in displacement of
the first agent (6) and the second agent (8) from the
multimerization reagent (4). This displacement of the first agent
(6) and second agent (8) in turn results in a dissociation of the
first agent (6) and the second agent (8) from the TCR/CD3 complex
and the accessory molecule CD28, thereby terminating the
stimulation/expansion of the cell population (2). Thus, as said
above, the present invention provides the advantage that the time
period of the stimulation or expansion of a T cell population can
be exactly controlled and therefore also the functional status of
the population of T cells can be closely controlled. After the
elution of the cells as illustrated in FIG. 1d, the first agent
(6), the second reagent (8) as well as the analogue (20) of free
first partner of the binding partner C1 and the second free partner
of the binding partner C2 can be easily removed from the stimulated
cell population (2) via a "removal cartridge" described in
International patent application WO 2013/124474. In addition, and
importantly, in case the initial sample was a population of
lymphocytes, for example, in form of PMBCs obtained from a Ficoll
gradient, the T cell population (2) is now available for serial
expansion as defined here. Since the expanded cell population (e.g.
by an initial stimulation via CD3/CD28) can be transfected during
expansion e.g. with a T cell receptor (TCR) or a chimeric antigen
receptor (CAR, also known as artificial T cell receptor), the
genetically modified cells can then be liberated from the initial
stimulus and subsequently be stimulated with a second type of
stimulus e.g. via the de novo introduced receptor. These second
stimuli may comprise an antigenic stimulus in form of a peptide/MHC
molecule, the cognate (cross-linking) ligand of the genetically
introduced receptor (e.g. natural ligand of a CAR) or any ligand
(such as an antibody) that directly binds within the framework of
the new receptor (e.g. by recognizing constant regions within the
receptor). Thus, the T cell population obtained from this serial
expansion can be used for adoptive cell transfer.
[0071] FIG. 3 shows a further embodiment of an expansion method of
the invention. Also the sample used in this Example comprises a
population of T cells (2) that carry two specific cell surface
molecules (30) and (32), with the cell surface molecule (30) being
a TCR/CD3 complex and the cell surface molecule (32) being the
accessory molecule CD28. In FIG. 3a the population of T cells (2)
is shown after being contacted with a multimerization reagent (4).
Also in this Example, the multimerization reagent (4) has
reversibly immobilized thereon (bound thereto) as first agent (6)
an anti-CD3 antibody or an antigen binding fragment thereof that
provides a primary activation signal to the T cells and as second
agent (8) an anti-CD28 antibody or an antigen binding fragment
thereof that stimulates CD28 as accessory molecule.
[0072] The multimerization reagent (4) shown in the Example of FIG.
3 comprises only one type binding site Z1 (42) for the reversible
binding of both the first agent (6) and the second agent (8). Both
the first agent (6) and the second agent (8) comprise at least one
binding partner C1 (6a, 8a), wherein both the binding partner C1
(6a) and the binding partner (8a) are able of reversibly binding to
the binding site Z1 (44) of the multimerization reagent. Thus, for
immobilization, the first agent (6) and the second agent (8),
respectively are bound to the multimerization reagent (4) via the
reversible bond formed between the binding partner C1 (6a) and the
binding partner C2 and the binding site Z1 (42). The binding
partners C1 and C2 can either be different or identical. For
example, the binding partner C1 can be a streptavidin binding
peptide of the sequences Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID
NO: 01), the "Strep-tag.RTM.") while the binding partner C2 can be
the streptavidin binding peptide of the sequence
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).sub.3-Trp-Ser-His-Pro-Gln--
Phe-Glu-Lys ((SEQ ID NO: 04), also known as "di-tag3")) or of the
sequence
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer).sub.2-Trp-Ser-His-Pro-Gln--
Phe-Glu-Lys ((SEQ ID NO: 05), also known as "the di-tag2"),
described by Junttila et al., Proteomics 5 (2005), 1199-1203 or
U.S. Pat. No. 7,981,632). All these streptavidin binding peptides
bind to the same binding site, namely the biotin binding site of
streptavidin. If one or more of such streptavidin binding peptides
is used as binding partners C1 and C2, the multimerization reagent
(4) is a streptavidin mutein. As shown in FIG. 3, a soluble
multimerization reagent (4) is used. In the case of a streptavidin
mutein, this soluble multimerization reagent may, for example, be
an oligomer or a polymer of streptavidin or avidin or of any mutein
(analog) of streptavidin or avidin. The oligomer may comprise three
or more monomers of streptavidin, avidin or a mutein therof. The
oligomer or polymer may be crosslinked by a polysaccharide. Such
oligomers or polymers of streptavidin or of avidin or of muteins of
streptavidin or of avidin can in a first step be prepared by the
introduction of carboxyl residues into a polysaccharide, e. g.
dextran, essentially as described in "Noguchi, A., Takahashi,
[0073] T., Yamaguchi, T., Kitamura, K., Takakura, Y., Hashida, M.
& Sezaki, H. (1992). Preparation and properties of the
immunoconjugate composed of anti-human colon cancer monoclonal
antibody and mitomycin C dextran conjugate. Bioconjugate Chemistry
3,132-137". In a second step, streptavidin or avidin or muteins
thereof are coupled via primary amino groups of internal lysine
residue and/or the free N-terminus to the carboxyl groups in the
dextran backbone using conventional carbodiimide chemistry.
Alternatively, cross-linked oligomers or polymers of streptavidin
or avidin or of any muten of streptavidin or avidin may also be
obtained by crosslinking via bifunctional linkers such as
glutardialdehyde or by other methods described in the
literature.
[0074] Using as binding partners C1 and C2, moieties that bind to
the same binding site (42) of the multimerization agent has the
advantage that, as shown in FIG. 3b, the same free partner (of the
first binding partner C1 and also of the second binding partner C2)
or analogue thereof can be used to terminate the expansion of the
population of T cells (2) and to release this population of T cells
(2) from the multimerization agent. In the Example of FIG. 3, an
analogue of the first and second partner C1 and C2 such as biotin
or a biotin derivate (iminobiotin or desthiobiotin) can be
conveniently used for the termination of the expansion and the
elution of the population of T cells (2).
[0075] As shown in FIG. 3c, after the elution of the cells as
illustrated in FIG. 1d, the first agent (6), the second reagent (8)
as well as biotin as the analogue (20) of free first partner of the
binding partner C1 and the second free partner of the binding
partner C2 can be easily removed from the stimulated cell
population (2) via a "removal cartridge" described in International
patent application WO 2013/124474. In addition, the embodiment of
using a soluble multimerization reagent (4) has the further
advantage of being able to avoid any solid support such as magnetic
beads. This means there is no risk of contamination of the
activated T cells by such magnetic beads. This also means that a
process that is compliant with GMP standards can be much easier
established compared to the known method such as the use of
Dynabeads.RTM. in which additional measures have to be taken to
ensure that the final expanded T cell population is free of
magnetic beads. Furthermore, the use of a soluble multimerisation
agent makes it much easier to remove the same from the activated
cell population (T cells, B cells or also natural killer cells)
since the cells can be simple sedimented by centrifugation and the
supernatant including the soluble multimerization agent can be
discarded. Alternatively, the soluble multimerization agent can be
removed from the expanded cell population in a gel permeations
matrix of the removal cartridge of International patent application
WO 2013/124474. Since no solid phase (e.g. magnetic beads) are
present, the present invention also provides for an automated
closed system for expansion of the cells that can be integrated
into known cell expansion systems such as the Xuri Cell Expansion
System W25 and WAVE Bioreactor 2/10 System, available from GE
Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) or
the Quantum.RTM. Cell Expansion System, available from TerumoBCT
Inc. (Lakewood, Colo., USA).
[0076] FIG. 4 shows the results of an experiment in which CD3+ T
responder cells were proliferated after being stimulated in vitro
with .alpha.CD3 and .alpha.CD28 Fab fragments that were reversibly
immobilized on beads coated with the streptavidin mutein
Strep-tactin.RTM.. FIG. 4A in a histogram showing size-distribution
(forward scatter) of stimulated cells, FIG. 4B depicts histograms
representing the degree of proliferation according to the number of
cells per cell division that are indicated on top of FIG. 4B (0
represents undivided cells; 5 represents cells that have gone
through at least 5 divisions), and FIG. 4C shows a picture of the
culture dish after 4 days of stimulation.
[0077] FIG. 5 shows the results of the differential intracellular
calcium mobilization in Jurkat cells that are either labelled with
the .alpha.CD3 antibody OKT3 or with Fab fragments of OKT3 being
multimerized with Strep-tactin.RTM. (also referred to as Fab
multimers herein). FIG. 5A: Jurkat cells were loaded with the
calcium-sensitive dye Indo-1-AM and calcium release was triggered
by injection of either .alpha.CD3 mAb (black squares) or .alpha.CD3
OKT3 Fab multimers (derived from the parental cell line OKT3) with
or without prior D-biotin disruption (dark grey triangles and light
grey circles respectively) compared to injection of PBS (inverted
white triangles). Application of ionomycine served as positive
control. Time-resolved changes in intracellular Ca.sup.2+
concentration were monitored by flow-cytometry based on the change
in FL6/FL7 ratio. FIG. 5B: Indo-1-AM-labeled Jurkat cells were
activated by different .alpha.CD3 stimuli as described in FIG. 4a;
OKT3: upper graph and .alpha.CD3 Fab-multimer: middle graph)
followed by subsequent (t=1405) D-biotin mediated disruption of
.alpha.CD3 Fab-multimer signaling. Injection of PBS (lower graph)
and ionomycine served as negative or positive control. Data are
representative of three different experiments.
[0078] FIG. 6 shows the result of the reversible staining of cells
by anti CD3 OKT3 Fab-multimers. Freshly isolated PBMCs were stained
with either a monoclonal antibody (left dot plot, parental clone
for the Fab-multimers) or cognate PE-labeled Fab-multimers and
analyzed either before (second left column) or after treatment with
D-biotin (middle column). Remaining Fab monomers were then detected
after subsequent washing steps using fresh PE-labeled
Strep-Tactin.RTM. (second right column). Secondary Fab-multimer
staining of reversibly stained cells served as control (right
column). Only live (PI.sup.negative) cells are shown. Numbers in
dot plots indicate the percentage of cells within gates.
[0079] FIG. 7 shows the isolation of cells by reversible binding of
anti-CD28 Fab fragments multimerized with Strep-Tactin.RTM. labeled
with phycoerythrine as a fluorescent label. CD28+ cells were
selected/isolation by Fab-multimer magnetic cell selection from
freshly isolated PMBCs as described in International Patent
Application WO2013/011011. Before selection cells were control
stained with either the cognate fluorescent .alpha.CD28-multimers
(left dot plot) or with an antibody directed against the
immunoglobulin kappa light chain (second left dot plot, .alpha.-Ig
kappa mAb). After selection, cells were treated with D-biotin and
subsequently washed to remove magnetic beads and Fab-monomers.
Liberated CD28+ cells were subsequently (re-)stained either with
CD28 Fab-multimers (second right dot plot) or with the .alpha.-Ig
kappa mAb (right dot plot) to detect potentially remaining
Fab-monomers. Only live (PI.sup.negative) CD3+ cells are shown.
Numbers in dot plots indicate the percentage of cells within
gates.
[0080] FIG. 8 shows the results of an experiment in which CD3+ T
responder cells were proliferated after being stimulated in vitro
with reversible .alpha.CD3/.alpha.CD28 Fab fragments that were
reversibly immobilized on soluble oligomeric Strep-tactin.RTM.
acting a soluble multimerization reagent. For the experiments the
results of which are shown in FIG. 8, 300.000 CD3+ responder T
cells (Tresp) were labeled with 2 .mu.M Carboxyfluorescein
succinimidyl ester (CFSE) and stimulated with varying amounts of a
preparation of soluble Streptactin oligomers on which a combination
of .alpha.CD3 Fab fragment and .alpha.CD28 Fab both carrying a
Strep-tag as streptavidin binding peptide at the heavy chain were
immobilized. ("1.times." corresponds to 3 .mu.g multimerized
Strep-tactin functionalized with 0.5.mu.g .alpha.CD3- and 0.5 .mu.g
.alpha.CD28 Fab; numbers indicate fold amount of "1.times."). Tresp
cells either left unstimulated or were stimulated with blank
Strep-tactin multimers (no Fab) served as negative control. Tresp
cells were seeded in duplicates in 48-well plates along with
300.000 CD3 negative autologous feeder cells (irradiated with 30Gy)
in lml cell culture medium supplemented with 20 U/ml interleukin 2
(IL-2). Cells were incubated at 37.degree. C. without media
exchange and proliferation was analyzed according to CFSE dilution
after 5 days by FACS analysis (FIG. 8B). FIG. 8A shows size
distribution of cells after 5 days in culture. Histograms show live
CD3+ cells, while FIG. 8C shows cells after culture that were
liberated by stimulation reagents after treated with 1 mM D-biotin
and washed. The dissociation and removal of monomeric Fab fragments
was analyzed by restaining with Strep-Tactin.RTM. labeled with
phycoerythrine (ST-PE) as a fluorescent label and a representative
histogram is shown. FIG. 8D shows the absolute number of live
(trypan blue negative) cells after 5 days was counted using a
Neubauer counting chamber and plotted against the respective
stimulation condition. Median cell numbers are shown in FIG. 8D;
error bars indicate standard deviation (SD). FIG. 8E shows a
picture of the culture dish after 5 days of stimulation.
[0081] FIG. 9 depicts an illustration of the serial expansion
method of the present invention (FIG. 9a) while FIG. 9b briefly
describes some features and advantages of the serial expansion.
[0082] FIG. 10 shows an arrangement of the invention that can be
used together with the expansion methods of the invention. This
arrangement (100) includes a bioreactor (50), a first "removal
cartridge" (70) and a second "removal cartridge" (90). The
bioreactor (50) is fluidly connected to the first removable
cartridge (70), and the first removal cartridge is fluidly
connected to the second removal cartridge (90). This arrangement
(100) can be part of a device for automated cell expansion and
purification as described here.
[0083] In the bioreactor (50) an expansion method as described
herein is carried out, for example an expansion method illustrated
in FIG. 3 that makes use of a soluble multimerization reagent. In
this case, after termination of the activation/expansion of the
cell population (2) by addition of a competitor (20) (free partner
of the binding partner C1 or an analogue thereof) the reaction
mixtures that is released from the bioreactor contains the expanded
population of cells (2), the first agent (6), the second agent (8)
as well as the soluble multimerization reagent (4). In this
example, the first agent (6) is a CD3 binding antibody fragment
that includes a streptavidin binding peptide as binding partner C1,
the second agent (8) is a CD28 binding antibody fragment that
includes a streptavidin binding peptide as binding partner C1 and
the competitor (20) (free analogue of the binding partner C1) is
biotin. This reaction mixture is applied on the first removal
cartridge (70). This first removal cartridge (70) is a removal
cartridge as described in
[0084] International patent application WO 2013/124474 that
includes a chromatography column with a suitable stationary phase.
The stationary phase can serve both an affinity chromatography
matrix and, at the same time, can act as gel permeation matrix.
This affinity chromatography matrix has an affinity reagent
immobilized thereon. The affinity reagent may, in the case of the
current Example, for instance, be streptavidin, a streptavidin
mutein, avidin, an avidin mutein or a mixture thereof. Thus, the
first agent (6) and the second agent (8) bind to the affinity
reagent via their streptavidin binding peptide. Also biotin as the
competitor (20) binds to the affinity reagent. Thus, these three
reagents are all being immobilized on the chromatography matrix of
the first removal cartridge while the expanded cell population (2)
and the soluble multimerization reagent (4) pass through the
stationary phase. This "flow through" is then applied onto the
second removal cartridge (90). Also this second removal cartridge
(90) comprises a stationary phase. This stationary phase comprises
a second affinity reagent thereon which is able to bind to the
binding site Z1 (42) of the multimerization reagent (4). This
affinity reagent may for example be biotin that is covalently bound
to the stationary phase. Such a stationary phase may, for example,
be d-biotin Sepharose.TM. obtainable from Affiland S.A. (Ans-Liege,
Belgium). Thus, the soluble multimerization reagent (4) will be
bound (retained) on the stationary phase of the second removal
cartridge (90) while the expanded population of cells (2) passes
through the stationary phase and is being freed of any reactants.
The population of cells (2) is now in a condition for any further
use, for example, for diagnostic applications (for example, further
FACS.TM. sorting) or for any cell based therapeutic application. It
is noted here that it is of course also possible to change the
order of the first "removal cartridge" (70) and the second "removal
cartridge" (90) in an arrangement (100), such that bioreactor (50)
is (directly) fluidly connected to the second removable cartridge
(90), and the first removal cartridge (70) is arranged after and
fluidly connected to the second removal cartridge (90). In this
arrangement the multimerization reagent (4) will first be removed
from the population of cells (2) and subsequently the first agent
(6), the second (8) and e.g. the competitor (20) are removed. Such
an arrangement is also encompassed in the present invention and can
also be part of a device for automated cell expansion and
purification as described here.
[0085] FIG. 11 shows a further embodiment of an arrangement of the
invention that can be used together with the expansion methods of
the invention. This arrangement (110) includes a bioreactor (50), a
first "removal cartridge" (70) and a second "removal cartridge"
(90). The bioreactor (50) is fluidly connected to the first
removable cartridge (70), and the first removal cartridge is
fluidly connected to the second removal cartridge (90). In
addition, the second removal cartridge (110) is fluidly connected
to the bioreactor (50). This arrangement (110) can also be part of
a device for automated cell expansion and purification as described
here. When used, for example, together with an expansion method
that employs a soluble multimerization reagent (4), a purified
expanded population of cells (2) is obtained as eluate of the
second removal cartridge (90). Since the removal cartridge (90) is
fluidly connected to the bioreactor (50), the population of cells
(2) can be transferred back into the bioreactor (50), for example,
to serial clonal expansion as described here, by transfecting the
population of cells, for example, with the gene of an T cell
receptor and subsequent further (second) expansion using an
expansion method of the invention.
[0086] FIG. 12 shows a further embodiment of an arrangement of the
invention that can be used together with the expansion methods of
the invention. This arrangement (120) includes a bioreactor (50), a
first "removal cartridge" (70) and a second "removal cartridge"
(90). The bioreactor (50) is fluidly connected to the first
removable cartridge (70), and the first removal cartridge is
fluidly connected to the second removal cartridge (90). Similar to
the embodiment shown in FIG. 11, the second removal cartridge (110)
is fluidly connected to the bioreactor (50). However, a "selection
cartridge" (92) as described in International patent application WO
2013/124474 is arranged in between the second removal cartridge
(90) and the bioreactor (50). Thus, a subpopulation of cells (2a)
that is comprised in the population of cells (2) can be
selected/enriched via this "selection cartridge" (92) as described
in WO 2013/124474. This subpopulation of cells (2a) can either be
transferred into the bioreactor (50), for example, to undergo
serial expansion as described here. Alternatively (not shown), this
subpopulation of cells (2a) can be used for cell based therapy. It
is again noted here that the use of a soluble multimerization
reagent as described here allows the design of automated cell
purification and expansion devices which are functionally closed
and thus not prone to contamination. In addition, since soluble
multimerization reagent avoids the need for solid phase materials
such as magnetic beads, such cell purification devices can be
designed as continuous flow devices.
[0087] FIG. 13 shows the expansion kinetics of proliferation of
purified CD4+ and CD8+ T responder cells (Tresp) that were
stimulated in vitro either with .alpha.CD3/.alpha.CD28 Fab
fragments or with .alpha.CD3/.alpha.CD28/.alpha.CD8 that were
reversibly immobilized on two kinds of a soluble oligomeric
Strep-tactin.RTM. mutein acting as soluble multimerization reagent.
The first kind of oligomeric Strep-tactin.RTM. was the fraction of
the oligomeric streptavidin mutein (n.gtoreq.3) obtained in Example
5 (also referred herein as "conventional Streptactin.RTM.
backbone", illustrated by the triangle symbol with the tip down in
FIG. 13), the second kind of this oligomeric streptavidin mutein
used as soluble multimerization reagent was an oligomer that was
obtained by reacting the soluble oligomeric streptavidin mutein
with biotinylated human serum albumin (HSA) This HSA based soluble
multimerization reagent is also referred herein as "large
Streptactin.RTM. backbone). In the experiments of FIG. 13 the
expansion was carried out without medium exchange. The results for
the CD4+ T responder cells are shown in FIG. 13A, the results for
the CD8+ T responder cells are shown in FIG. 13B. In this context,
it is noted that the experimentally used soluble multimerization
reagents that were functionalized by reversibly binding first
agents, and optionally second and third agents are referred to in
the Figures as "Streptamer.RTM. multimers"
[0088] FIG. 14 shows the expansion kinetics of proliferation of
purified CD4+ and CD8+ T responder cells (Tresp) that were
stimulated in vitro with .alpha.CD3/.alpha.CD28 Fab fragments that
were reversibly immobilized fragments that were reversibly
immobilized with two kinds of soluble oligomeric Strep-tactin.RTM.
acting as soluble multimerization reagent. The first kind of
oligomeric Strep-tactin.RTM. was the fraction of the oligomeric
streptavidin mutein (n.gtoreq.3) obtained in Example 5 (also
referred herein as "conventional Streptactin.RTM. backbone",
illustrated by the triangle symbol with the tip on top in FIG. 14),
the second kind of this oligomeric streptavidin mutein used as
soluble multimerization reagent was the HSA based soluble
multimerization agent, the above-mentioned "large Streptactin.RTM.
backbone"). In the experiments of FIG. 14 the expansion was carried
out with medium exchange. The results for the CD4+ T responder
cells are shown in FIG. 14, the results for the CD8+ T responder
cells are shown in FIG. 14B.
[0089] FIG. 15 shows the combined data from the results obtained in
FIGS. 13 and 14 for the expansion kinetics of proliferation of
purified CD4+ and CD8+ T responder cells, with FIG. 15A depicting
the results for CD4+ T cells and FIG. 15B depicting the results for
the CD8+ T cells. Straight lines are used for the culturing with
medium exchange on day 3, while dashed lines depict the values
obtained for the degree of expansion without media exchange on day
3. The data shown in FIG. 15 are normalized on the input cell
number. Only data for the Tresp stimulated with the oligomeric
streptavidin mutein (n.gtoreq.3), the Tresp stimulated with the
commercially available Dynabeads (positive control) and the
unstimulated T cells (negative control) are shown but no data on
the multimerization reagent with the "large Streptactin.RTM.
backbone".
[0090] FIG. 16 shows early cluster formation of T cells after
activation of purified CD4+ and CD8+ T responder cells stimulated
in vitro with .alpha.CD3/.alpha.CD28 Fab fragments that were
reversibly immobilized on the soluble oligomeric streptavidin
mutein (n.gtoreq.3) described in Example 5. FIG. 16A depicts the
results for CD4+ T cells and FIG. 16B depicts the results for the
CD8+ T cells. Data for the Tresp stimulated with the soluble
multimerization reagent (the oligomeric streptavidin mutein), the
Tresp stimulated with the commercially available Dynabeads
(positive control) and the unstimulated T cells (negative control)
are shown.
[0091] FIG. 17 shows the expansion kinetics and phenotype of CD3+
central memory T cells (Tcm) (CD3+CD62L+CD45RA- Tcm) polyclonally
stimulated in vitro with .alpha.CD3/.alpha.CD28 Fab fragments that
were reversibly immobilized on the soluble oligomeric streptavidin
mutein (with n.gtoreq.3) described in Example 5. The graphs shown
in FIG. 17 represent the degree of proliferation according to the
number of cells harvested per time point, with FIG. 17A showing the
proliferation in only IL-2 supplemented media and in FIG. 17B
showing the proliferation in IL-2 and IL-15 supplemented media.
FIG. 17C shows a flow-cytometric analysis of CD62L and
[0092] CD127 surface expression after 14 days of culture in these
variable cytokine milieus.
[0093] FIG. 18 shows the kinetics of selective antigen-specific
(Ag-specific) expansion out of a bulk population of purified
CD3+CD62L+CD45RA- Tcm responder cells that were stimulated in vitro
with both a peptide:MHC molecule complex (that acts as first agent
that provides a primary activation signal to the cells) and
.alpha.CD28 Fab fragment (that acts as second agent that binds the
accessory molecule on the surface of the cells) and unstimulated T
cells (negative control) are shown. Both, the complex of
antigen-specific peptide with the MHC molecule and the .alpha.CD28
Fab fragment were reversibly immobilized on the same soluble
oligomeric streptavidin mutein (with n.gtoreq.3) described in
Example 5. The peptide used for the antigen-specific expansion in
FIG. 18A was the peptide CRVLCCYVL (SEQ ID NO: 06), amino acids
309-317 of the immediate-early 1 protein restricted by the HLA-C702
MHC molecule (described in Ameres et al, PLOS Pathogens, May 2013,
vol. 9, issue 5, e1003383) representing an HLA-C7/IE-1 epitope that
is specific for cytomegalovirus (CMV). The MHC I molecule that
presents the peptide carries at its C-terminus of the heavy chain
the streptavidin binding peptide
(SAWSHPQFEK(GGGS).sub.2GGSAWSHPQFEK (SEQ ID NO: 07), that is
commercially available as "Twin-Strep-tag.RTM." from IBA GmbH,
Gottingen, Germany) FIG. 18A shows exemplary flow-cytometric
analysis for the fraction of the Ag-specific cells that were
proliferated using the peptide:MHC-I complex specific for this
HLA-C7/IE-1 epitope as first agent that provides a primary
activation signal to the cells reversibly immobilized on the
soluble oligomeric streptavidin mutein. The graphs in FIG. 18B to
FIG. 18E illustrates the expansion kinetics of further
Ag-specificities according to the number of specific peptide:MHCI
multimer-positive cells harvested per time point in analogy to FIG.
18A using distinct complexes of an antigen-specific peptide with
the MHC I molecule as first agent that provides a primary
activation signal to the cells reversibly immobilized on the
soluble oligomeric streptavidin mutein. In more detail, FIG. 18B
shows the expansion of Ag-specific cells that were expanded using
the peptide:MHC-I complex specific for the pp65 epitope of CMV
(amino acids 341-350 (QYDPVAALF, (SEQ ID NO: 08)) restricted by
HLA-A2402), FIG. 18C shows the expansion of Ag-specific cells that
were expanded using another peptide:MHC-I complex specific for the
pp65 epitope of CMV (amino acids 265-274 RPHERNGFTV, (SEQ ID NO:
09)) restricted by HLA-B702), FIG. 18D shows the expansion of
Ag-specific cells that were proliferated using the peptide:MHC-I
complex specific for the hexon 5 epitope of adenovirus (amino acids
114-124 (CPYSGTAYNSL, (SEQ ID NO: 10)) restricted by HLA-B702),
FIG. 18E shows the expansion of Ag-specific cells that were
proliferated using the peptide:MHC-I complex specific for the
HLA-B7/IE-1.sub.309-317 epitope of CMV (exemplary FACS data see
above FIG. 18A). All peptide:MHC molecules bearing the Twin
Strep.RTM.-Tag are commercially available from IbaGmbH. In this
context, the amino acid sequences of the HLA-A*2402, HLA-B*0702 and
HLA-C*0702 molecules that carry the "Twin-Strep-tag.RTM." as their
C-terminus are shown as SEQ ID NO: 21, 22 and 23 in the
accompanying Sequence Listings, while the amino acid sequence of
the .beta..sub.2 microglobulin (which forms together with the a
chain, that means the HLA encoded molecules the respective MHC I
molecule) is shown as SEQ ID NO: 24 in the accompanying Sequence
Listing. In addition, FIG. 18F show exemplary flow-cytometric
analysis of CD62L and CD127 surface expression after 14 days of
culture for HLA-B7/Hexon5.sub.114-124 stimulated/expanded cells
from FIG. 18D.
[0094] FIG. 19 shows the kinetics of selective Ag-specific
expansion out of purified CD3+CD62L+CD45RA- Tcm responder cells
that were stimulated in vitro with a) antigen specific peptide MHC
I complexes and b) .alpha.CD28 Fab fragments that were reversibly
immobilized as first and second agent on soluble oligomeric
streptavidin muteins. For this purpose 500.000 CD3+CD62L+CD45RA-
responder Tcm cells (Tresp) were stimulated Ag-specifically using 3
.mu.l of a preparation of Streptactin multimerization reagent
functionalized with 0.5 .mu.g peptide:MHC class I complexes
equipped with a streptavidin binding peptide (the specific peptide
represents amino acids 114-124 (CPYSGTAYNSL, SEQ ID NO: 10) of the
Hexon 5 protein of the adenovirus restricted by HLA-B0702, see
above) and 0.5 .mu.g .alpha.CD28 Fab. As an alternative, 4.5 .mu.l
of a preparation of Streptactin multimerization reagent loaded with
0.5 .mu.g this peptide:MHC class I complex, 0.5 .mu.g .alpha.CD8
Fab and 0.5 .mu.g .alpha.CD28 Fab. For comparison, polyclonal
stimulation was performed, using 3 .mu.l of a preparation of
Streptactin multimerization reagent (1 mg/ml) either loaded with a
combination of 0.5 .mu.g .alpha.CD3 Fab and 0.5 .mu.g .alpha.CD28
Fab. Again as the alternative stimulation condition described
above, 4.5 .mu.l of a preparation of Streptactin multimers loaded
with 0.5 .mu.g .alpha.CD3 Fab, 0.5 .mu.g .alpha.CD8 Fab and 0.5
.mu.g .alpha.CD28 Fab was used. Untreated (unstimulated) Tresp
cells served as negative control and Tresp cells stimulated
polyclonal with Dynabeads as positive control. Tresp cells were
seeded in 48-well plates in 1 ml cell culture medium supplemented
with 30 U/ml IL-2 and 5ng/ml IL-15. Cells were incubated at
37.degree. C. with media exchange every 3 days and cell count was
analyzed after 7 and 14 days. The photographs shown in FIG. 19
represent the degree of cluster formation on day 5 for Ag-specific
stimulation as exemplified for the HLA-B7/Hexon 5 epitope of
adenovirus.
[0095] FIG. 20 shows the yield and phenotype of expansion of
purified CD8+ T responder cells stimulated in vitro with
.alpha.CD3/.alpha.CD28 Fab fragments that were reversibly
immobilized on two kinds of soluble oligomeric Strep-tactin.RTM.
acting a soluble multimerization reagent. The first kind of
oligomeric Strep-tactin.RTM. was the fraction of the oligomeric
streptavidin mutein (nmericobtained in Example 5 (conventional
backbone), the second kind of this oligomeric streptavidin mutein
used as soluble multimerization reagent was the soluble oligomer
described above and referred herein as "large" Streptactin.RTM.
backbone. In these experiments, the fraction of the oligomeric
conventional streptavidin mutein (n.gtoreq.3) was also used as a
multimerization reagent that were either functionalized with single
Fab fragments (third bar in FIG. 20A and FIG. 20B) or with a
combination of .alpha.CD3 and .alpha.CD28 Fab-fragments.
Furthermore to the combined stimulation with .alpha.CD3/.alpha.CD28
Fab fragments, also an additional .alpha.CD8 Fab fragment
(commercially available from IBA GmbH, Gottingen, Germany) was
immobilized in order to test whether it is possible to
preferentially stimulate a specific T cell subpopulation. FIG. 20A
shows a graph of bars that represent the degree of proliferation
according to the number of cells harvested at day 6 compared to the
negative controls (unstimulated purified CD8+ T responder cells)
and normalized to the positive control (purified CD8+ T responder
stimulated with commercially available Dynabeads (beads on which
.alpha.CD3 and .alpha.CD28 monoclonal antibodies are irreversible
immobilized). FIG. 20B shows flow-cytometric analysis of the
surface expression of CD8 and the T cell surface molecule CD45RO
(that is indicative of T cell proliferation and activation) after
cell culture. The various stimulating conditions were compared
using one-way ANOVA and no significant difference (n.s.) was
detected.
[0096] FIG. 21 shows the yield and phenotype for the expansion of
purified CD8+ T responder cells stimulated in vitro with
.alpha.CD3/.alpha.CD28 Fab fragments that were reversibly
immobilized on soluble oligomeric Strep-tactin.RTM. acting as a
soluble multimerization reagent that were either functionalized
with single Fab fragments or with a combination of Fab-fragments
(as already described above). In these experiments, the CD8+ T
responder cells were stimulated with the soluble multimerization
reagent (the soluble oligomeric Strep-tactin.RTM. (1 mg/ml) of
Example 5) which was functionalized with varying amounts of
.alpha.CD3 and .alpha.CD28 Fab fragments, optionally together with
the .alpha.CD8 Fab fragment described above. The term "1.times."
corresponds to 1.5 .mu.g multimerized Streptactin functionalized
with 0.5 .mu.g .alpha.CD3 Fab fragment alone and 1.5 .mu.g
multimerized Streptactin functionalized with 0.5 .mu.g .alpha.CD28
Fab alone), or 3 .mu.l of a preparation of oligomeric Streptactin
loaded with 0.5 .mu.g .alpha.CD3 Fab fragment and 0.5 .mu.g
.alpha.CD28 Fab, or 4.5 .mu.l of a preparation of Streptactin
multimers loaded with 0.5 .mu.g strep-tagged .alpha.CD3, 0.5 .mu.g
strep-tagged .alpha.CD8 and 0.5 .mu.g strep-tagged .alpha.CD28 Fab.
Accordingly, the term "2.times." corresponds to 3.0 .mu.g
multimerized Streptactin functionalized with 1 .mu.g .alpha.CD3 Fab
fragment alone and 3.0 .mu.g multimerized Streptactin
functionalized with 1 .mu.g .alpha.CD28 Fab alone, meaning that
twice the amount of immobilized .alpha.CD3 Fab fragment was used.
Untreated Tresp cells served as negative control and purified CD8+
T responder stimulated with commercially available Dynabeads (beads
on which .alpha.CD3 and .alpha.CD28 monoclonal antibodies are
irreversible immobilized) as positive control. FIG. 21A shows a
graphs in which the bars represent the degree of proliferation
according to the number of cells harvested at day 5 compared to the
negative controls and normalized to the positive control. FIG. 21B
shows FACS analysis of CD8 and CD45RO surface expression after cell
culture.
[0097] FIG. 22 shows the activation of intracellular signaling
cascades of transduced Jurkat cells that have been modified to
express an .alpha.CD19 chimeric antigen receptor (CAR), and that
were stimulated using the oligomeric Strep-tactin.RTM. of Example 5
as soluble multimerization reagent. The specificity of a CAR is
typically derived from a scFv region assembled from the
antigen-binding region of a monoclonal antibody (mAb) that
specifically binds a target/tumor associated antigen such as CD19
and links it to T cell specific signaling (described in Hudecek et
al, Clin Cancer Res. 2013 Jun. 15; 19 (12): 3153-3164. In the
experiments the extracellular domain (ECD) of CD19, which contains
the natural ligand of the .quadrature.CD19 CAR as well as the
polyclonal .alpha.IgG F(ab).sub.2 fragment that recognizes the IgG4
spacer (donkey-anti-human F(ab).sub.2 is commercially available
from Jackson Immuno Research) within the .alpha.CD19-CAR were also
used in this experiment as first agent that provides a primary
activation signal to the jurkat cells. The reversibly
immobilization to the soluble oligomeric streptavidin mutein was
provided by the streptavidin peptide
SAWSHPQFEK(GGGS).sub.2GGSAWSHPQFEK (SEQ ID NO: 07) that was fused
to the C-terminus of the ECD of CD19 or by the biotinylated
(Fab).sub.2 fragment of the .alpha.IgG (since the streptavidin
mutein "m2" binds biotin with reduced affinity, this binding is
reversible and can for example be displaced by addition of an
excess of free biotin). In the control experiment of FIG. 22A
300.000 CD3+ Jurkat responder cells (Jresp) were stimulated with
varying amounts of a mixture of preparations of oligomeric
Streptactin (1 mg/ml) that was functionalized with the .alpha.CD3
Fab and the .alpha.CD28 Fab ("x1" corresponds to 3 .mu.g
multimerized Streptactin functionalized with 0.5 .mu.g .alpha.CD3-
and 0.5 .mu.g .alpha.CD28 Fab--polyclonal Streptamer multimer). In
the experiment of FIG. 22B 3 .mu.l of a preparation of the
oligomeric Streptactin was functionalized with 0.5 .mu.g (x1) or 1
.mu.g (x2) of the extracellular domain (ECD) of CD19 or with 3
.mu.l of a preparation of the oligomeric Streptactin loaded with
0.5 .mu.g (x1) or 1 .mu.g (x2) .alpha.IgG that recognizes the IgG4
spacer (which are both CAR-specific Streptamer.RTM. multimers).
Jresp stimulated with Dynabeads (beads on which .alpha.CD3 and
.alpha.CD28 monoclonal antibodies are irreversible immobilized) or
PMA and ionomycin served as positive controls. Jresp cells were
seeded in 1.5 ml Eppendorf tubes in 200 .mu.lcell culture medium
supplemented with 30 U/ml IL-2. Cells were incubated at 37.degree.
C. and put on ice and lysed after 0 min to 20 min of
stimulation.
[0098] FIG. 23 shows the expansion of purified CD3.sup.+ T
responder cells stimulated in vitro with .alpha.CD3/.alpha.CD28 Fab
fragments that were reversibly immobilized on the soluble
oligomeric Strep-tactin.RTM. of Example 5 that served a soluble
multimerization reagent. In one experiment, in addition to
.alpha.CD3/.alpha.CD28 Fab fragments, also an .alpha.CD8 Fab
fragment commercially available from IBA GmbH, Gottingen, Germany
(catalogue number 6-8000-203) was immobilized on the soluble
oligomer of the streptavidin mutein in order to test whether it is
possible to preferentially stimulate in vitro the CD8+ T cell
subpopulation within the bulk CD3.sup.+ culture with a
multimerization reagent of the invention having reversibly
immobilized thereon also an .alpha.CD8 Fab fragment. In more
detail, 500.000 purified CD3.sup.+ responder T cells (Tresp) were
stimulated with 3 .mu.l of a preparation of oligomeric Streptavidin
(1 mg/ml) loaded with a combination of 0.5 .mu.g of the .alpha.CD3
and 0.5 .mu.g of the .alpha.CD28 Fab. As an alternative approach,
4.5 .mu.l of the Streptactin oligomer were loaded with 0.5 .mu.g
.alpha.CD3, 0.5 .mu.g .alpha.CD8 Fab and 0.5 .mu.g .alpha.CD28 Fab
described above. Unstimulated Tresp cells served as negative
control and Tresp stimulated with Dynabeads (beads on which
.alpha.CD3 and .alpha.CD28 monoclonal antibodies are irreversible
immobilized) served as positive control.
[0099] FIG. 24 depicts exemplary strategies for the generation of
oligomeric streptavidin muteins that can be used as soluble
multimerization reagent of the invention. FIG. 24A shows that in a
first step, the streptavidin mutein "m2" (SAm2) that comprises the
amino acid sequence lle.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47
(SEQ ID NO: 03) at sequence positions 44 to 47 of wild type
streptavidin is used for generation of oligomeric streptavidin
muteins having a "conventional backbone". In a second step,
oligomeric soluble streptavidin muteins having a "large backbone"
can be generated by either by coupling of streptavidin mutein with
biotinylated carrier protein such as human serum albumin (HSA) or
by coupling the streptavidin muteins with synthetic carriers such
as PEG. FIG. 24B: Biotinylation of human serum albumin (HSA).
DETAILED DESCRIPTION OF THE INVENTION
[0100] The present invention provides methods, kits and an
apparatus for expanding a population of cells or for inducing a
population of T cells to proliferate.
[0101] The term "population of cells" as used herein encompasses
all cells which can be expanded by binding to a cell surface
receptor a first agent that provides a primary activation signal to
the cells. It is also possible that for expansion of the population
of cells, binding of second agent to a second cell surface receptor
(accessory molecule) might be needed to produce a co-stimulatory
signal required for expansion of the cells. In some embodiments the
cell population may be a population of lymphocytes including, but
not limited a population of B cells, a population of T cells or a
population of natural killer cells. Illustrative examples of cell
populations are B cells carrying CD40 or CD137 (both cell
population can be proliferated upon binding of only a first agent
that provides an activation signal, for example 4-1BB ligand; or an
.alpha.CD40 antibody molecule or an .alpha.CD137 antibody molecule
(see for example Zhang et al., 2010, J Immunol, 184:787-795)).
Other illustrative examples for agents (either first or second)
that may be used for the expansion of B cells are agents that bind
to IgG, CD19, CD28 or CD14, for example .alpha.CD19, .alpha.IgG,
.alpha.CD28, or .alpha.CD14 antibody molecules. It is also
envisioned that first or second agents for the expansion of B cell
may comprise ligands for toll like receptors or interleukins, such
as IL-21 (see for example Dienz O, et al. 2009. J. Exp. Med.
206:69). It is noted that lipopolysaccharide dependent activation
of B cells is also encompassed in the present invention, as a
lipopolysaccharide can also be used as first agent and can be
equipped with a binding partner C1 as used herein. Other
illustrative examples of suitable cell populations include T cell
population that expand after being activated by binding of a first
agent to TCR/CD3 and binding of a second agent to an accessory
molecule on the T cell such as CD28. In this case, the first agent
stimulates a TCR/CD3 complex-associated signal in the T cells and
the second agent provides a secondary stimulus by binding CD28 as
accessory molecule. Agents that can be used for the expansion of T
cells may also include interleukins, such as IL-2, IL-7, IL-15, or
IL-21 (see for example Cornish et al. 2006, Blood. 108 (2):600-8,
Bazdar and Sieg, 2007, Journal of Virology, 2007, 81
(22):12670-12674, Battalia et al, 2013, Immunology, 139
(1):109-120). Other illustrative examples for agents that may be
used for the expansion of T cells are agents that bind to CD8, CD45
or CD90, such as .alpha.CD8, .alpha.CD45 or .alpha.CD90 antibodies.
Illustrative examples of T cell population including
antigen-specific T cells, T helper cells, cytotoxic T cells, memory
T cell (an illustrative example of memory T-cells are
CD62L.sup.+CD8.sup.+ specific central memory T cells) or regulatory
T cells (an illustrative example of Treg are
CD4.sup.+CD25.sup.+CD45RA.sup.+ Treg cells). The term "T cell
(population)" as used herein also includes T cells that comprise a
chimeric antigen receptor (CAR) that is also known as artificial T
cell receptors or chimeric T cell receptors. Thus, a T cell
population that comprises a chimeric antigen receptor can also be
expanded using the methods, reagents and devices of the present
invention. See in this respect also Example 15 in which Jurkat
cells that express a chimeric CD19 specific antigen receptor (CAR)
were stimulated using a soluble multimerization reagent of the
present invention. Another illustrative example of a suitable cell
population includes natural killer cells (NK cells), which may for
example be expanded with agents that bind to CD16 or CD56, such as
for example .alpha.CD16 or .alpha.CD56 antibodies. In illustrative
example for such an .alpha.CD16 antibody is the antibody 3G8 with a
VH sequence set forth in SEQ ID NO: 25 and a VL sequence set forth
in SEQ ID NO: 26 (see for example Hoshino et al, Blood. 1991 Dec.
15; 78 (12):3232-40.). Another agent that may be used for expansion
of NK cells may be IL-15 (see for example Vitale et al. 2002. The
Anatomical Record. 266:87-92). Yet another illustrative example of
a suitable cell population includes monocytes, which may for
instance be expanded using an agent that binds to CD14, such as an
.alpha.CD14 antibody molecule. The cell population can be of any
mammalian origin, including but not limited to human, rabbit,
guinea pig, squirrel, hamster, cat, dog, lemur, goat, pig, horse,
rhesus monkey, macaque, or a chimpanzee.
[0102] Thus, in line with the above, this invention pertains to
methods for selectively inducing ex vivo expansion of a population
of cells such as B cells, T cells or natural killer cells in the
absence of exogenous growth factors, such as lymphokines, and
accessory cells. In addition, the proliferation of these cells such
as B cells or T cells can be induced without the need for antigen,
thus providing an expanded cell population such as a T cell
population which is polyclonal with respect to antigen reactivity.
The methods disclosed herein may provide for sustained
proliferation of a selected population of T cells such as CD4+ or
CD8+ T cells over an extended period of time to yield a multi-fold
increase in the number of these cells relative to the original T
cell population. In general, in case of a (clonal) expansion of a
lymphocyte population as described herein, all progeny may share
the same antigen specificity as the cell population that was
selected for expansion.
[0103] Also in line with the above, provided by this invention are
methods for expanding a population of antigen specific T cells. To
produce a population of antigen specific T cells, T cells are
contacted with an antigen in a form suitable to trigger a primary
activation signal in the T cell, i.e., the antigen is presented to
the T cell such that a signal is triggered in the T cell through
the TCR/CD3 complex. For example, the antigen can be presented to
the T cell by an antigen presenting cell in conjunction with an MHC
molecule. An antigen presenting cell, such as a B cell, macrophage,
monocyte, dendritic cell, Langerhans cell, or other cell which can
present antigen to a T cell, can be incubated with the T cell in
the presence of the antigen (e.g., a soluble antigen) such that the
antigen presenting cell presents the antigen to the T cell.
Alternatively, a cell expressing an antigen of interest can be
incubated with the T cell. For example, a tumor cell expressing
tumor-associated antigens can be incubated with a T cell together
to induce a tumor-specific response. Similarly, a cell infected
with a pathogen, e.g., a virus, which presents antigens of the
pathogen can be incubated with a T cell. Following antigen specific
activation of a population of T cells, the cells can be expanded in
accordance with the methods of the invention. For example, after
antigen specificity has been established, T cells can be expanded
by culture with an anti-CD3 antibody (used as first agent) and an
anti-CD28 antibody (used as second agent) according to the methods
described herein. In another embodiment, the first agent can be an
MHC I: peptide complex, which binds to an antigen specific T cell
population. In such an embodiment, any antigen specific peptide
that is known and that can be complexed with the respective MHC I
molecule can be used. See in this respect Examples 11 and 12 in
which selective Antigen-specific expansion of Tcm responder cells
out of bulk CD330 central memory T cells was exemplied for four
different antigen-specific cells. Alternatively, it is also
possible to use as first agent the natural ligand of a receptor
that triggers of cell expansion. See in this respect Example 15 in
which the extracellular domain of CD19 caused the activation of
intracellular signaling cascades of transduced Jurkat cells that
were modified to express chimeric CD19 binding antigen receptor
(CAR).
[0104] The sample of the cell population can be from any suitable
source, typically all sample of a body tissue or a body fluid such
as blood. In the latter case, the sample might for example, be a
population of peripheral blood mononucleated cells (PBMC) that can
be obtained by standard isolation methods such a ficoll gradient of
blood cells. The cell population to be expanded can however also be
in purified form and might have been isolated using an reversible
cell staining/isolation technology as described patent in U.S. Pat.
Nos. 7,776,562, 8,298,782, International Patent application
WO02/054065 or International Patent Application WO2013/011011.
Alternatively, the population of cells can also be obtained by cell
sorting via negative magnetic immunoadherence as described in U.S.
Pat. No. 6,352,694 B1 or European Patent EP 0 700 430 B1. If an
isolation method described here is used in basic research, the
sample might be cells of in vitro cell culture experiments. The
sample will typically have been prepared in form of a fluid, such
as a solution or dispersion.
[0105] In line with the above, in one embodiment the invention
provides an in vitro-method of expanding a population of cells,
comprising contacting a sample comprising a population of cells
with a multimerization reagent. The multimerization reagent has
reversibly immobilized thereon (bound thereto) a first agent that
provides a primary activation signal to the cells, wherein the
multimerisation reagent comprising at least one binding site Z1 for
the reversible binding of the first agent. The first agent
comprises at least one binding partner C1, wherein the binding
partner C1 is able of reversibly binding to the binding site Z1 of
the multimerization reagent, wherein the first agent is bound to
the multimerization reagent via the reversible bond formed between
the binding partner C1 and the binding site Z1. The first agent
binds to a receptor molecule on the surface of the cells, thereby
providing a primary activation signal to the cells and thereby
activating the cells.
[0106] In another embodiment, the invention provides a method,
wherein the multimerization agent has reversibly immobilized
thereon (bound thereto) a second agent that stimulates an accessory
molecule on the surface of the cells. The second agent comprises a
binding partner C2, wherein the binding partner C2 is able of being
reversibly bound to a binding site Z2 of the multimerization
reagent, wherein the second agent is bound to the multimerization
reagent via the reversible bond formed between the binding partner
C2 and the binding site Z2. The second agent binds to the accessory
molecule on the surface on the surface of the cells, thereby
stimulating the activated cells. In this embodiment the first agent
may stimulate a TCR/CD3 complex-associated signal in the T cells
and may be a binding agent that specifically binds CD3. In this
embodiment the accessory molecule on the T cell may be CD28 and the
second agent that binds the accessory molecule is a binding reagent
that specifically binds CD28. In this case, the first agent that
specifically binds CD3 may be selected from the group consisting of
an anti-CD3-antibody, a divalent antibody fragment of an anti-CD3
antibody, a monovalent antibody fragment of an anti-CD3-antibody,
and a proteinaceous CD3 binding molecule with antibody-like binding
properties. Also the second agent that specifically binds CD28 may
be selected from the group consisting of an anti-CD28-antibody, a
divalent antibody fragment of an anti-CD28 antibody, a monovalent
antibody fragment of an anti-CD28-antibody, and a proteinaceous
CD28 binding molecule with antibody-like binding properties. The
divalent antibody fragment may be an (Fab).sub.2' -fragment, or a
divalent single-chain Fv fragment while the monovalent antibody
fragment may be selected from the group consisting of a Fab
fragment, a Fv fragment, and a single-chain Fv fragment (scFv). A
proteinaceous CD3 or CD28 binding molecule with antibody-like
binding properties may be an aptamer, a mutein based on a
polypeptide of the lipocalin family, a glubody, a protein based on
the ankyrin scaffold, a protein based on the crystalline scaffold,
an adnectin, and an avimer.
[0107] In general the first and the second agent that is used in
the present invention may, for instance be, an antibody, a fragment
thereof and a proteinaceous binding molecule with antibody-like
functions. Examples of (recombinant) antibody fragments are Fab
fragments, Fv fragments, single-chain Fv fragments (scFv), a
divalent antibody fragment such as an (Fab)2'-fragment, diabodies,
triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441),
decabodies (Stone, E., et al., Journal of Immunological Methods
(2007) 318, 88-94) and other domain antibodies (Holt, L. J., et
al., Trends Biotechnol. (2003), 21, 11, 484-490). In some
embodiments one or more binding sites of the first or second agent
may be a bivalent proteinaceous artificial binding molecule such as
a dimeric lipocalin mutein that is also known as "duocalin". In
some embodiments the receptor binding reagent may have a single
second binding site, i.e., it may be monovalent. Examples of
monovalent first or second agents include, but are not limited to,
a monovalent antibody fragment, a proteinaceous binding molecule
with antibody-like binding properties or an MHC molecule. Examples
of monovalent antibody fragments include, but are not limited to a
Fab fragment, a Fv fragment, and a single-chain Fv fragment (scFv),
including a divalent single-chain Fv fragment.
[0108] As mentioned above, an example of a proteinaceous binding
molecule with antibody-like functions is a mutein based on a
polypeptide of the lipocalin family (see for example, WO 03/029462,
Beste et al., Proc. Natl. Acad. Sci. U.S.A. (1999) 96, 1898-1903).
Lipocalins, such as the bilin binding protein, the human neutrophil
gelatinase-associated lipocalin, human Apolipoprotein D or human
tear lipocalin possess natural ligand-binding sites that can be
modified so that they bind a given target. Further examples of a
proteinaceous binding molecule with antibody-like binding
properties that can be used as a receptor binding reagent that
specifically binds to the receptor molecule include, but are not
limited to, the so-called glubodies (see e.g. international patent
application WO 96/23879), proteins based on the ankyrin scaffold
(Mosavi, L. K., et al., Protein Science (2004) 13, 6, 1435-1448) or
crystalline scaffold (e.g. international patent application WO
01/04144) the proteins described in Skerra, J. Mol. Recognit.
(2000) 13, 167-187, AdNectins, tetranectins and avimers. Avimers,
including multivalent avimer proteins evolved by exon shuffling of
a family of human receptor domains, contain so called A-domains
that occur as strings of multiple domains in several cell surface
receptors (Silverman, J., et al., Nature Biotechnology (2005) 23,
1556-1561). Adnectins, derived from a domain of human fibronectin,
contain three loops that can be engineered for immunoglobulin-like
binding to targets (Gill, D. S. & Damle, N. K., Current Opinion
in Biotechnology (2006) 17, 653-658). Tetranectins, derived from
the respective human homotrimeric protein, likewise contain loop
regions in a C-type lectin domain that can be engineered for
desired binding (ibid.). Peptoids, which can act as protein
ligands, are oligo(N-alkyl) glycines that differ from peptides in
that the side chain is connected to the amide nitrogen rather than
the a carbon atom. Peptoids are typically resistant to proteases
and other modifying enzymes and can have a much higher cell
permeability than peptides (see e.g. Kwon, Y.-U., and Kodadek, T.,
J. Am. Chem. Soc. (2007) 129, 1508-1509). Yet further examples of
suitable proteinaceous binding molecules are an EGF-like domain, a
Kringle-domain, a fibronectin type I domain, a fibronectin type II
domain, a fibronectin type III domain, a PAN domain, a Gla domain,
a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain,
tendamistat, a Kazal-type serine protease inhibitor domain, a
Trefoil (P-type) domain, a von Willebrand factor type C domain, an
Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I
repeat, LDL-receptor class A domain, a Sushi domain, a Link domain,
a Thrombospondin type I domain, an immunoglobulin domain or a an
immunoglobulin-like domain (for example, domain antibodies or camel
heavy chain antibodies), a C-type lectin domain, a MAM domain, a
von Willebrand factor type A domain, a Somatomedin B domain, a
WAP-type four disulfide core domain, a F5/8 type C domain, a
Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-type
EGF-like domain, a C2 domain, "Kappabodies" (cf. Ill. et al.,
Protein Eng (1997) 10, 949-57, a so called "minibody" (Martin et
al., EMBO J (1994) 13, 5303-5309), a diabody (cf. Holliger et al.,
PNAS USA (1993) 90, 6444-6448), a so called "Janusis" (cf.
Traunecker et al., EMBO J (1991) 10, 3655-3659, or Traunecker et
al., Int J Cancer (1992) Suppl 7, 51-52), a nanobody, a microbody,
an affilin, an affibody, a knottin, ubiquitin, a zinc-finger
protein, an autofluorescent protein or a leucine-rich repeat
protein. An example of a nucleic acid molecule with antibody-like
functions is an aptamer. An aptamer folds into a defined
three-dimensional motif and shows high affinity for a given target
structure.
[0109] Turning now the multimerization reagent, the binding sites
Z1 and Z2 of the multimerization agent can be identical (see also
the Example of FIG. 3). In this case, a single multimerization
agent may be used.
[0110] In the embodiment that a reversibly bond first and,
optionally second agent is used, the multimerization reagent may be
immobilized on a solid surface. Any solid surface (support) can be
used for the immobilization of the multimerization reagent.
Illustrative examples of solid surfaces on which the
multimerization reagent can be immobilized include a magnetic bead,
a polymeric bead, a cell culture plate, a microtiter plate, a
membrane, or a hollow fiber. Hollow fibers are, for example, used
as bioreactor in the Quantum.RTM. Cell Expansion System, available
from TerumoBCT Inc. (Lakewood, Colo., USA). The multimerization
reagent is usually covalently attached to the solid support,
however, non-covalent interactions can also be used for
immobilization, for example on plastic substrates, if wanted. As
also explained in more detail below, the multimerization reagent
can, for example, be a streptavidin or avidin mutein that
reversibly binds a streptavidin binding peptide. Such streptavidin
muteins can be covalently attached to any surface, for example,
resin (beads) used for chromatography purification and are
commercially available in such form from IBA GmbH, Gottingen, for
example, as Strep-Tactin.RTM. Sepharose, Strep-Tactin.RTM.
Superflow.RTM., Strep-Tactin.RTM. Superflow.RTM. high capacity or
Strep-Tactin.RTM. MacroPrep.RTM.. Other illustrative examples
multimerization reagents that are readily commercially available
are immobilized metal affinity chromatography (IMAC) resins such as
the TALON.RTM. resins (Westburg, Leusden, The Netherlands) that can
be used for the reversible immobilization of oligo-histidine tagged
(his-tagged) proteins in general, meaning here, for the reversible
binding of a first or a second agent that carries as first binding
partner C1 or second binding partner C2 an oligohistidine tag such
as an penta- or hexa-histidine tag. Other examples of
multimerzation reagents are calmodulin sepharose available from GE
Life Sciences which can be used together with a first or second
agent that comprises a calmodulin binding peptide as binding
partner C1 or C2 or sepharose, to which glutathion is coupled. In
the case, the binding partner C1 or C2 is
glutathion-S-transferase.
[0111] In other embodiments of the method of the invention the
multimerization reagent can be in a soluble form. In principle, the
same multimerization agents can be used as in the case of a
multimerization reagent that is immobilized on a solid support. The
multimerization reagent is soluble form, can for example, be a
streptavidin mutein oligomer, a calmodulin oligomer, a compound
(oligomer) that provides least two chelating groups K, wherein the
at least two chelating groups are capable of binding to a
transition metal ion, thereby rendering moiety A capable of binding
to an oligohistidine affinity tag, multimeric
glutathione-S-transferase, or a biotinylated carrier protein.
[0112] As explained above, the first and second agent has, in
addition to the binding site that is able to bind the respective
cell surface receptor molecule, a binding partner C1 or C2 (which
will be referred to as "binding partner C" in the following for the
ease of reference). This binding partner C is able to bind to a
binding site Z of the multimerization reagent (Z means either
binding site Z1 or binding site Z2 of the multimerization reagent)
C. The non-covalent bond that is formed between the binding partner
C that is included in the first or second agent and the binding
site(s) Z of the multimerization reagent may be of any desired
strength and affinity, as long as it is disruptable or reversible
under the conditions under which the method of the invention is
performed. The dissociation constant (KD) of the binding between
the binding partner C that is included in the receptor binding
reagent and the binding site Z of the multimerization reagent may
have a value in the range from about 10.sup.--2 M to about
10.sup.--13 M. Thus, this reversible bond can, for example, have a
K.sub.D from about 10.sup.--2 M to about 10.sup.--13 M, or from
about 10.sup.--3 M to about 10.sup.--12 M or from about 10.sup.--4
M to about 10.sup.--11M, or from about 10.sup.--5 M to about
10.sup.--10 M. The K.sub.D of this bond as well as the K.sub.D,
k.sub.off and k.sub.on rate of the bond formed between the binding
site B of the receptor binding reagent and the receptor molecule
can be determined by any suitable means, for example, by
fluorescence titration, equilibrium dialysis or surface plasmon
resonance. The receptor molecule binding reagent may include at
least one, including two, three or more, second binding partners C
and the affinity reagent may include at least two, such as three,
four, five, six, seven, eight or more binding sites for the binding
partner that is included in the receptor molecule binding reagent.
As described in U.S. Pat. Nos. 7,776,562, 8,298,782 or
International Patent application WO 2002/054065 any combination of
a binding partner C and an affinity agent with one or more
corresponding binding sites Z can be chosen, as long as the binding
partner C and the binding site Z of the affinity agent are able to
reversibly bind or multimerize in a (multivalent) complex, which
typically goes along with an avidity effect.
[0113] The binding partner included in the first or second agent
may be an oligopeptide, a polypeptide, a protein, a nucleic acid, a
lipid, a saccharide, an oligosaccharide, or a polysaccharide. Such
a binding partner has a higher affinity to the binding site of the
multimerization reagent than to other matter. Examples of a
respective binding partner include, but are not limited to, an
immunoglobulin molecule, a fragment thereof and a proteinaceous
binding molecule with antibody-like functions.
[0114] In some embodiments the binding partner C that is included
in the first or second agent includes biotin and the affinity
reagent includes a streptavidin analogue or an avidin analogue that
reversibly binds to biotin.
[0115] In some embodiments the binding partner C that is included
in the first or second agent includes a biotin analogue that
reversibly binds to streptavidin or avidin, and the affinity
reagent includes streptavidin, avidin, a streptavidin analogue or
an avidin analogue that reversibly binds to the respective biotin
analogue.
[0116] In some further embodiments the binding partner C that is
included in the first or second agent includes a streptavidin or
avidin binding peptide and the affinity reagent includes
streptavidin, avidin, a streptavidin analogue or an avidin analogue
that reversibly binds to the respective streptavidin or avidin
binding peptide.
[0117] In some embodiments the binding partner that is included in
the first or second agent may include a streptavidin-binding
peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 01) and the
affinity reagent may include a streptavidin mutein (analogue) that
comprise the amino acid sequence
Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 (SEQ ID NO: 02) at
sequence positions 44 to 47 of wild type streptavidin or the
streptavidin mutein (analogue) that comprises the amino acid
sequence lle.sup.44-Gly45-Ala.sup.46-Arg.sup.47 (SEQ ID NO: 03) at
sequence positons 44 to 47 of wild type streptavidin, both of which
are described in U.S. Pat. No. 6,103,493, for example, and are
commercially available under the trademark Strep-Tactin.RTM.. The
streptavidin binding peptides might, for example, be single
peptides such as the "Strep-tag.RTM." described in U.S. Pat. No.
5,506,121, for example, or streptavidin binding peptides having a
sequential arrangement of two or more individual binding modules as
described in International Patent Publication WO 02/077018 or U.S.
Pat. No. 7,981,632.
[0118] In some embodiment the binding partner C of the first or
second agent includes a moiety known to the skilled artisan as an
affinity tag. In such an embodiment the affinity reagent includes a
corresponding binding partner, for example, an antibody or an
antibody fragment, known to bind to the affinity tag. As a few
illustrative examples of known affinity tags, the binding partner
that is included in the first or second agent may include an
oligohistidine, an immunoglobulin domain, maltose-binding protein,
glutathione-S-transferase (GST), chitin binding protein (CBP) or
thioredoxin, calmodulin binding peptide (CBP), FLAG'-peptide, the
HA-tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, (SEQ ID NO:
11)), the VSV-G-tag (sequence:
Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys, (SEQ ID NO: 12)), the
HSV-tag (sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp,
(SEQ ID NO: 13)), the T7 epitope
[0119] (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly, (SEQ ID NO: 14)),
maltose binding protein (MBP), the HSV epitope of the sequence
Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 13) of
herpes simplex virus glycoprotein D, the "myc" epitope of the
transcription factor c-myc of the sequence
Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO: 15), the V5-tag
(sequence: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr,
SEQ ID NO: 16), or glutathione-S-transferase (GST). In such an
embodiment the complex formed between the one or more binding sites
of the multimerisation reagent, in this case an antibody or
antibody fragment, and the antigen can be disrupted competitively
by adding the free antigen, i.e. the free peptide (epitope tag) or
the free protein (such as MBP or CBP). The affinity tag might also
be an oligonucleotide tag. Such an oligonucleotide tag may, for
instance, be used to hybridize to an oligonucleotide with a
complementary sequence, linked to or included in the affinity
reagent.
[0120] In some embodiments the binding between the binding partner
C that is included in the first or second agent and one or more
binding sites of the multimerization reagent occurs in the presence
of a divalent, a trivalent or a tetravalent cation. In this regard
in some embodiments the multimerization reagent includes a
divalent, a trivalent or a tetravalent cation, typically held, e.g.
complexed, by means of a suitable chelator. The binding partner
that is included in the receptor binding reagent may in such an
embodiment include a moiety that includes, e.g. complexes, a
divalent, a trivalent or a tetravalent cation. Examples of a
respective metal chelator, include, but are not limited to,
ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethylene
glycol tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid
(DTPA), N,N-bis(carboxymethyl)glycine (also called nitrilotriacetic
acid, NTA), or 1,2-bis(o-aminophen-oxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA). As an example, EDTA forms a complex with most
monovalent, divalent, trivalent and tetravalent metal ions, such as
e.g. calcium (Ca.sup.2+), manganese (Mn.sup.2+), copper
(Cu.sup.2+), iron (Fe.sup.2+), cobalt (Co.sup.3+) and zirconium
(Zr.sup.4+), while BAPTA is specific for Ca.sup.2+. As an
illustrative example, a standard method used in the art is the
formation of a complex between an oligohistidine tag and copper
(Cu.sup.2+), nickel (Ni.sup.2+), cobalt (Co.sup.2+), or zinc
(Zn.sup.2+) ions, which are presented by means of the chelator
nitrilotriacetic acid (NTA).
[0121] In some embodiments the binding partner C that is included
in the first or second agent includes a calmodulin binding peptide
and the affinity reagent includes multimeric calmodulin as
described in U.S. Pat. No. 5,985,658 or as described herein with
reference to FIG. 2, for example. In some embodiments the binding
partner C that is included in the first or second agent includes a
FLAG peptide and the affinity reagent includes an antibody that
binds to the
[0122] FLAG peptide, e.g. the FLAG peptide, which binds to the
monoclonal antibody 4E11 as described in U.S. Pat. No. 4,851,341.
In one embodiment the binding partner C that is included in the
first or second agent includes an oligohistidine tag and the
affinity reagent includes an antibody or a transition metal ion
binding the oligohistidine tag. The disruption of all these binding
complexes may be accomplished by metal ion chelation, e.g. calcium
chelation, for instance by adding EDTA or EGTA (supra). Calmodulin,
antibodies such as 4E11 or chelated metal ions or free chelators
may be multimerized by conventional methods, e.g. by biotinylation
and complexation with streptavidin or avidin or multimers thereof
or by the introduction of carboxyl residues into a polysaccharide,
e.g. dextran, essentially as described in Noguchi, A, et al.
Bioconjugate Chemistry (1992) 3, 132-137 in a first step and
linking calmodulin or antibodies or chelated metal ions or free
chelators via primary amino groups to the carboxyl groups in the
polysaccharide, e.g. dextran, backbone using conventional
carbodiimide chemistry in a second step. In such embodiments the
binding between the binding partner C that is included in the first
or second agent and the one or more binding sites Z of the
multimerization reagent can be disrupted by metal ion chelation.
The metal chelation may, for example, be accomplished by addition
of EGTA or EDTA.
[0123] In some embodiments, in particular, if the multimerization
reagent is in soluble form and is based on streptavidin or avidin,
it is an oligomer or a polymer of streptavidin or avidin or of any
mutein (analogue) of streptavidin or avidin. The binding site Z is
the natural biotin binding of avidin or streptavidin. The
respective oligomer or polymer may be crosslinked by a
polysaccharide. In one embodiment oligomers or polymers of
streptavidin or of avidin or of muteins (analogs) of streptavidin
or of avidin are prepared by the introduction of carboxyl residues
into a polysaccharide, e. g. dextran, essentially as described in
Noguchi, A, et al., Bioconjugate Chemistry (1992) 3,132-137 in a
first step. Then streptavidin or avidin or analogues thereof may be
linked via primary amino groups of internal lysine residue and/or
the free N-terminus to the carboxyl groups in the dextran backbone
using conventional carbodiimide chemistry in a second step. In
addition, cross-linked oligomers or polymers of streptavidin or
avidin or of any mutein (analogue) of streptavidin or avidin may
also be obtained by crosslinking individual streptavidin or avidin
molecules (the tetrameric homodimer of streptavidin or avidin is
referred herein as an "individual molecule" or smallest building
block of a respective oligomer or polymer) via bifunctional
molecules, serving as a linker, such as glutardialdehyde or by
other methods described in the art. It is, for example, possible to
generate oligomers of streptavidin muteins by introducing, in a
first step, thiol groups into the streptavidin mutein (this can,
for example, be done by reaction the streptavidin mutein
2-iminothiolan (Trauts reagent) and by activating, in a separate
reaction amino groups available in the streptavidin mutein. This
activation of amino groups can be achieved by reaction of the
streptavidin mutein with a commercially available
heterobifunctional crosslinkers such as sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo SMCC) or
Succinimidyl-6-maleimidopropionamido)hexanoate (SMPH). In a second
step, the two reaction products so obtained are mixed together,
leading to the reaction of the thiol groups contained in the one
batch of modified streptavidin mutein with the activated (by
maleimid functions) amino acids of the other batch of modified
streptavidin mutein. By this reaction, multimers/oligomers of the
streptavidin mutein are formed. These oligomers can have any
suitable number of "individual molecule" or streptavidin building
block" higher than 3 and the oligomerization degree can be varied
according to the reaction condition (see FIG. 24). After reacting
these two batches of the modified streptavidin mutein, the
oligmeric soluble multimerization reagent is typically isolated via
size exclusion chromatography and any desired fraction can be used
as multimerization reagent. Typically, the oligomers do not have
(and do not need to have) a single molecular weight but they
usually observe a statistical weight distribution such as Gaussian
distribution. Any oligomer with more than three streptavidin
homotetramers (building blocks; (n.gtoreq.3)) can be used as
soluble multimerization reagent. The oligomers might have, for
example from 3 to 25 steptavidin mutein homotetramers. With a
molecular weight of about 50 kDa for streptavidin muteins such as
the mutein "m1" or "m2" described in more detail below, these
soluble oligomers have a molecular weight from about 150 kDa to
about 1250 kDa. Since each streptavidin molecule/mutein has four
biotin binding sites such a multimerization reagent provides 12 to
100 binding sites Z1 (and Z2) as described herein.
[0124] In accordance with the above disclosure, in addition to such
oligomeric multimerization reagents that only contain cross-linked
streptavidin homotetramers, it is possible to react tetrameric
streptavidin muteins to a carrier to obtain multimerization
reagents that are used in the present invention. In addition to the
above described reaction with a polysaccharide, it is also possible
to use physiologically or pharmaceutically acceptable proteins such
as serum albumin (for example human serum albumin (HSA) or bovine
serum albumin (BSA) as carrier protein. In such a case, the
streptavidin mutein (either as individual homo-tetramer or also in
the form of oligomers with n.gtoreq.3) can be coupled to the
carrier protein via non-covalent interaction. For this purpose,
biotinylated BSA (which is commercially available from various
suppliers such as ThermoFisher Scientific, Sigma Aldrich or
Vectorlabs, to name only a few) can be reacted with the
streptavidin mutein. When so doing, some of the streptavidin
oligomers will non-covalently bind via one or more biotin binding
sites (Z1, Z2) to the biotinylated carrier protein, leaving the
majority of the binding sites (Z1, Z2) of the oligomer available
for binding the agents such as the first agent and optionally the
second agent and any further agent as described herein. Thus, by
such an approach a soluble multimerization reagent with a multitude
of binding sites Z1 can be conveniently prepared (see FIG. 24).
Alternatively, a streptavidin mutein (either as individual
homo-tetramer or also in the form of oligomers with n.gtoreq.3) can
be covalently coupled to a synthetic carrier such as a polyethylene
glycol (PEG) molecule. Any suitable PEG molecule can be used for
this purpose, as long as the PEG molecule and the respective
multimerization reagent is soluble. Typically, PEG molecule up to a
molecular weight of 1000 Da are all soluble in water or culture
media that may be used in the present invention. Such PEG based
multimerization reagent can be easily prepared using commercially
available activated PEG molecules (for example, PEG-NHS derivatives
available from NOF North America Corporation, Irvine, Calif., USA,
or activated PEG derivatives available from Creative PEGWorks,
Chapel Hills, N.C., USA) with amino groups of the streptavidin
mutein.
[0125] Under streptavidin or wild-type streptavidin
(wt-streptavidin), the amino acid sequence disclosed by Argarana et
al., Nucleic Acids Res. 14 (1986) 1871-1882 is referred to.
Streptavidin muteins are polypeptides which are distinguished from
the sequence of wild-type streptavidin by one or more amino acid
substitutions, deletions or additions and which retain the binding
properties of wt-streptavidin. Streptavidin-like polypeptides and
streptavidin muteins are polypeptides which essentially are
immunologically equivalent to wild-type streptavidin and are in
particular capable of binding biotin, biotin derivative or biotin
analogues with the same or different affinity as wt-streptavidin.
Streptavidin-like polypeptides or streptavidin muteins may contain
amino acids which are not part of wild-type streptavidin or they
may include only a part of wild-type streptavidin.
Streptavidin-like polypeptides are also polypeptides which are not
identical to wild-type streptavidin, since the host does not have
the enzymes which are required in order to transform the
host-produced polypeptide into the structure of wild-type
streptavidin. The term "streptavidin" also includes streptavidin
tetramers and streptavidin dimers, in particular streptavidin
homotetramers, streptavidin homodimers, streptavidin
heterotetramers and strepavidin heterodimers. Each subunit normally
has a binding site for biotin or biotin analogues or for
streptavidin-binding peptides. Examples of streptavidins or
streptavidin muteins are mentioned, for example, in WO 86/02077, DE
19641876 A1, U.S. Pat. No. 6,022,951, WO 98/40396 or WO
96/24606.
[0126] In a preferred embodiment, streptavidin muteins that are
used as multimerization reagent are those streptavidin muteins
which are described in U.S. Pat. No. 6,103,493 and also in DE 196
41 876.3. These streptavidin muteins have at least one mutation
within the region of amino acid positions 44 to 53, based on the
amino acid sequence of wild-type streptavidin. Preference is given
to muteins of a minimal streptavidin, which start N-terminally in
the region of amino acids 10 to 16 of wild-type streptavidin and
end C-terminally in the region of amino acids 133 to 142 of
wild-type streptavidin. Examples of such preferred streptavidin
muteins have a hydrophobic aliphatic amino acid instead of Glu at
position 44, any amino acid at position 45, a hydrophobic aliphatic
amino acid at position 46 or/and a basic amino acid instead of Val
at position 47. The streptavidin mutein may be a mutein that
comprises the amino acid sequence
Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 (SEQ ID NO: 02) at
sequence positions 44 to 47 or the streptavidin mutein (analog)
that comprises the amino acid sequence
lle.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47 (SEQ ID NO: 03) at
sequence positions 44 to 47 of wild type streptavidin. Such muteins
are described in U.S. Pat. No. 6,103,493, for example, and are
commercially available from IBA GmbH in the form of mutein "m1" and
mutein "m2" under the trademark Strep-Tactin.RTM..
[0127] A method according to the present invention may in some
embodiments be used to deplete a sample of reagents that have
previously been used in cell expansion. The first or second agent
and the respective free partner (the competition agent) may, for
instance, be present included in the eluate of an expansion method
as described above. Using a method according to the invention such
reagents may be at least essentially, including entirely removed
from a sample, e.g. from a cell population. As an illustrative
example, a first or second agent as defined above may be depleted
from a sample to levels that are below the detection limit of e.g.
FACS or Western Blot. A competition reagent (free first or second
binding partner or analogue thereof) may have been used in order to
terminate and control the expansion and release the cell population
form the multimerization agent. This competition reagent may have a
binding site that is capable of specifically binding to the binding
site Z of the affinity reagent in a "removal cartridge" of WO
2013/124474. In such an embodiment the respective method of the
invention may also serve in depleting the first and second agent
and the competition reagent, including removing the same.
[0128] A method according to the present invention may be carried
out at any temperature at which the viability of the cell
population is at least essentially uncompromised. When reference is
made herein to conditions that are at least essentially not
harmful, not detrimental or at least essentially not compromising
viability, conditions are referred to, under which the percentage
of the population of cells that are to be expanded with full
viability, is at least 70%, including at least 75%, at least 80%,
at least 85%, at least 90%, at least 92%, at least 95%, at least
97%, at least 98%, at least 99% or at least 99.5%. In some
embodiments a method according to the invention is carried out at a
temperature of about 20.degree. C. or higher. Depending on the cell
population to be expanded a suitable temperature range may for
instance be from about 20.degree. C. to about 45.degree. C.,
including from about 25.degree. C. to about 40.degree. C., or from
about 32.degree. C. to 37.degree. C. In some embodiments a method
according to the invention is carried out at a constant temperature
value, or at a selected temperature value .+-.about 5.degree. C.,
.+-.about 4.degree. C., .+-.about 3.degree. C., .+-.about 2.degree.
C., .+-.about 1.degree. C. or .+-.about 0.5.degree. C. The person
skilled in the art is able to empirically determine a suitable
temperature, taking into account the nature of the cells and the
expansion conditions. Typically human cells are expanded at a
temperature such as 37.degree. C.
[0129] In a further embodiment, the invention provides an in
vitro-method of expanding a population of cells, comprising
contacting a sample comprising a population of cells with a
multimerization reagent, wherein the multimerization reagent is in
a soluble form and has immobilized thereon (bound thereto) a first
agent that provides a primary activation signal to the cells. The
multimerization reagent comprises at least one binding site Z1 for
the binding of the first agent, wherein the first agent comprises
at least one binding partner C1, wherein the binding partner C1 is
able of binding to the binding site Z1 of the multimerization
reagent. The first agent is bound to the multimerization reagent
via the bond formed between the binding partner C1 and the binding
site Z1, and the first agent binds to a receptor molecule on the
surface of the cells, thereby providing a primary activation signal
to the cells and thereby activating the cells. It is expressly
noted here that when a soluble multimerization agent is used, the
bond between the binding part C1 and the binding site Z1 does not
need to be reversible.
[0130] In an embodiment of this second the multimerization agent
has immobilized thereon (bound thereto) a second agent that
stimulates an accessory molecule on the surface of the cells,
wherein the second agent comprises a binding partner C2, wherein
the binding partner C2 is able of being bound to a binding site Z2
of the multimerization reagent. The second agent is bound to the
multimerization reagent via the bond formed between the binding
partner C2 and the binding site Z2, wherein the second agent binds
to the accessory molecule on the surface on the surface of the
cells, thereby stimulating the activated cells.
[0131] In one embodiment of this second method, the bond formed
between the binding partner C1 and the binding site Z1 may be
irreversible and/or also the bond formed between the binding
partner C2 and the binding site Z2 may be irreversible.
[0132] In a different embodiment of this second method, the bond
formed between the binding partner C1 and the binding site Z1 may
be reversible. Also the bond formed between the binding partner C2
and the binding site Z2 may be reversible. In this case, the
dissociation constant (Ka) for the reversible binding between said
binding site Z1 and said binding partner C1 and/or for the
reversible binding between said binding site Z2 and said binding
partner C2 may be in the range of 10.sup.-2 M to 10.sup.-13 M.
[0133] In this second method that is based on a soluble
multimerization reagent, the first and second reagent as well as
the multimerization reagent and all other reagents and cell
populations can otherwise be used in the same manner as disclosed
above for the method that makes use of reversible between the first
or second agent and the multimerization reagent.
[0134] The invention further provides a reagent kit for expanding a
population of cells, the kit comprising [0135] (i) a
multimerization reagent, wherein the multimerization reagent
comprises at least one binding site Z for the reversible binding of
a first agent, [0136] (ii) a first agent that binds to a receptor
molecule on the surface of the cells, thereby providing a primary
activation signal to the cells and thereby activating the cells,
wherein the first agent comprises at least one binding partner C1,
wherein the binding partner C1 is able of reversibly binding to a
binding site Z1 of the multimerization reagent, wherein the first
agent is bound to the multimerization reagent via the reversible
bond formed between the binding partner C1 and the binding site Z1,
and [0137] (iii) a second agent that stimulates an accessory
molecule on the surface of the cells, wherein the second agent
comprises a binding partner C2, wherein the binding partner C2 is
able of reversibly binding to a binding site Z2 of the
multimerization reagent, wherein the second agent is bound to the
multimerization reagent via the bond formed between the binding
partner C2 and the binding site Z2, wherein the second agent binds
to the accessory molecule on the surface on the surface of the
cells, thereby stimulating the activated cells.
[0138] The invention also provides a reagent kit for expanding a
population of cells, the kit comprising [0139] (i) a
multimerization reagent, wherein the multimerization reagent is in
soluble form and comprises at least one binding site Z for the
reversible binding of a first agent, [0140] (ii) a first agent that
binds to a receptor molecule on the surface of the cells, thereby
providing a primary activation signal to the cells and thereby
activating the cells, wherein the first agent comprises at least
one binding partner C1, wherein the binding partner C1 is able of
binding to a binding site Z1 of the multimerization reagent,
wherein the first agent is bound to the multimerization reagent via
the reversible bond formed between the binding partner C1 and the
binding site Z1.
[0141] This second reagent kit may further comprises (iii) a second
agent that stimulates an accessory molecule on the surface of the
cells, wherein the second agent comprises a binding partner C2,
wherein the binding partner C2 is able of binding to a binding site
Z2 of the multimerization reagent, wherein the second agent is
bound to the multimerization reagent via the bond formed between
the binding partner C2 and the binding site Z2.
[0142] A kit as disclosed herein is in particular used when the
population of cells is a lymphocyte population.
[0143] In accordance with the disclosure above, the invention also
provides novel multimerization reagents and novel composition
comprising multimerization reagents that care capable of expanding
a population of cells. Such a multimerization reagent that is
capable of expanding a population of cells is a multimerisation
reagent that is in soluble form and comprises at least one binding
site Z1 for the reversible binding of a first agent that provides a
primary activation signal to the cells, wherein the multimerization
reagent has reversibly immobilized thereon (bound thereto) said
first agent that provides a primary activation signal to the cells;
wherein the first agent comprises at least one binding partner C1,
wherein the binding partner C1 is able of reversibly binding to the
at least one binding site Z1 of the multimerization reagent,
wherein the first agent is bound to the multimerization reagent via
the reversible bond formed between the binding partner C1 and the
binding site Z1. It should be noted here that such a
multimerization agent can have immobilized thereon any of the first
agent that are described herein.
[0144] A multimerization reagent of the invention may further
comprise at least one binding site Z2 for the reversible binding of
a second agent that stimulates an accessory molecule on the surface
of the cells, wherein the multimerization reagent has reversibly
immobilized thereon (bound thereto) the second agent that
stimulates an accessory molecule on the surface of the cells,
wherein the second agent comprises a binding partner C2, wherein
the binding partner C2 is able of binding to the at least one
binding site Z2 of the multimerization reagent. In this embodiment
the second agent is bound to the multimerization reagent via the
bond formed between the binding partner C2 and the binding site
Z2.
[0145] Also in line with the disclosure given above, such a
multimerization reagent is capable of expanding a lymphocyte
population or a subpopulation contained in the lympocyte
population. The lymphocyte population to be expanded may any
suitable population, for example, a B cell population, a T cell
population, or a natural killer cell population. The T-cell
population may be an antigen-specific T cell population, a T helper
cell population, a cytotoxic T cell, a memory T cell, a regulatory
T cell, or a natural killer T cell population. Accordingly, in such
embodiments of the multimerization reagent the first agent is able
to stimulate a TCR/CD3 complex-associated signal in the T cells.
The first agent present in the multimerization reagent may thus be
binding reagent that specifically binds CD3, while the second agent
that binds the accessory molecule may be a binding agent that
specifically binds CD28 or CD137.
[0146] In embodiments of the multimerization reagent the first
agent that specifically binds CD3 may be an anti-CD3-antibody, a
divalent antibody fragment of an anti-CD3 antibody, a monovalent
antibody fragment of an anti-CD3-antibody, and/or a proteinaceous
CD3 binding molecule with antibody-like binding properties. In
these embodiments, the second agent that specifically binds CD28 or
CD137 may be an anti-CD28-antibody, a divalent antibody fragment of
an anti-CD28 antibody, a monovalent antibody fragment of an
anti-CD28-antibody, a proteinaceous CD28 binding molecule with
antibody-like binding properties, an anti-CD137-antibody, a
divalent antibody fragment of an anti-CD137 antibody, a monovalent
antibody fragment of an anti-CD137-antibody, a proteinaceous CD137
binding molecule with antibody-like binding properties, 4-1BB
ligand, and any mixture thereof. Thus, a multimerization reagent of
the invention can generally have immobilized thereon one kind of
first agent and a mixture of second agents, for example, an
anti-CD3 antibody as first agent and for example, an anti-CD28
antibody and 4-1BB ligand as (joint) second agents.
[0147] If the multimerization reagent is to be used for the
expansion of B cells, the first agent immobilized on the
multimerization reagent may be a binding reagent that specifically
binds CD40 or CD137. In accordance with the disclosure given
herein, in such embodiments the first binding reagent that
specifically binds CD40 or CD137 may be selected from an
anti-CD40-antibody, a divalent antibody fragment of an anti-CD40
antibody, a monovalent antibody fragment of an anti-CD40-antibody,
and a proteinaceous CD40 binding molecule with antibody-like
binding properties or an anti-CD137-antibody, a divalent antibody
fragment of an anti-CD137 antibody, a monovalent antibody fragment
of an anti-CD137-antibody, a proteinaceous CD137 binding molecule
with antibody-like binding properties, and CD40 ligand (CD154).
[0148] Also in accordance with the general disclosure of the
present invention, in the multimerization reagent as described
herein the binding sites Z1 and Z2 of the multimerization reagent
can be identical. As described above, such a multimerization
reagent may comprises an oligomer or a polymer of streptavidin, an
oligomer or a polymer of avidin, an oligomer or a polymer of an
analog of streptavidin that reversibly binds biotin, an oligomer or
a polymer of an analog avidin that reversibly bind biotin, a
reagent that comprises at least two chelating groups K, wherein the
at least two chelating groups are capable of binding to a
transition metal ion, thereby rendering the reagent capable of
binding to an oligohistidine affinity tag, multimeric
glutathione-S-transferase, multimeric calmodulin and a biotinylated
carrier protein.
[0149] A novel composition provided herein that is capable of
expanding a population of cells may comprise [0150] (i) a first
multimerization reagent, wherein the first multimerisation reagent
is in soluble form and comprises at least one binding site Z1 for
the reversible binding of a first agent that provides a primary
activation signal to the cells, wherein the first multimerization
reagent has reversibly immobilized thereon (bound thereto) said
first agent that provides a primary activation signal to the cells,
wherein the first agent comprises at least one binding partner C1,
wherein the binding partner C1 is able of reversibly binding to the
at least one binding site Z1 of the multimerization reagent,
wherein the first agent is bound to the multimerization reagent via
the reversible bond formed between the binding partner C1 and the
binding site Z1, and [0151] (ii) a second multimerization reagent,
wherein the second multimerization reagent is in soluble form and
comprises at least one binding site Z2 for the reversible binding
of a second agent that stimulates an accessory molecule on the
surface of the cells, wherein the multimerization reagent has
reversibly immobilized thereon (bound thereto) said second agent
that stimulates an accessory molecule on the surface of the cells,
wherein the second agent comprises a binding partner C2, wherein
the binding partner C2 is able of binding to the at least one
binding site Z2 of the multimerization reagent, wherein the second
agent is bound to the multimerization reagent via the bond formed
between the binding partner C2 and the binding site Z2.
[0152] Such a novel composition is, for example, the reaction
mixture used in Example 13, in which two separate multimerization
reagents were functionalized either with an .alpha.CD3 Fab fragment
alone or an .alpha.CD28 Fab fragment alone. It is noted in this
context, that such a composition was shown in Example 13 to have
the same expansion efficiency as a single multimerization reagent
on which both the first agent and the second agent are jointly
immobilized. Thus, the combined use of two or more multimerization
reagents being functionalized individually with only one type of
reagent (for example, one first or one second agent) is
functionally equivalent to using for the expansion one joint
multimerization reagent which has immobilized thereon both a first
agent and a second agent. In this context, it is also noted that a
multimerization reagent of the present invention be functionalized
with as many agents (for example, one, two, three, four or even
more agents) that are intended to be used for the expansion of a
selected cell population. A third or fourth agent may, for example,
for example provide a stimulus for the expansion of a desired
subpopulation of cells. See in this context, for instance, Example
13 in which a soluble multimerization reagents was reversibly
functionalized with three reagents, namely a .alpha.CD3 Fab
fragment as first reagent, a .alpha.CD28 Fab fragment as second
reagent and a .alpha.CD8 Fab fragment as third reagent to enrich
the subpopulation of CD8+ T cells in a sample of a population of
CD3+ T cells (lymphocyte). By using such a combinations of agents
that can all be reversibly immobilized on the same multimerization
reagent, the present invention allows for the possibility to
preferentially expand or selectively enrich any desired cell
(sub)population from an sample that, for example, comprises a
variety of different subpopulations. In this context, it is noted
that it however also possible to use for this purpose use three
different multimerization reagents, for example, a first
multimerization reagent that is functionalized with only a
.alpha.CD3 Fab fragment, a second multimerisation reagent that is
functionalized with a .alpha.CD28 Fab fragment and a third
multimerization reagent that is functionalized with a .alpha.CD8
Fab fragment. Likewise, it is possible to use only two different
multimerization reagents, a first multimerization reagent that is
functionalized with only a .alpha.CD3 Fab fragment and a second
multimerisation reagent that is functionalized with both a
.alpha.CD28 Fab fragment and a .alpha.CD8 Fab fragment.
Accordingly, the present invention allows to design any kind of
wanted expansion reagent in a modular fashion.
[0153] The invention also provides an in vitro-method of serially
expanding a population of lymphocytes, wherein the population of
lymphocytes comprises T cells. This method comprises [0154]
contacting a sample comprising the T cell comprising population of
lymphocytes with a multimerization reagent, [0155] wherein the
multimerization reagent is in a soluble form and has reversibly
immobilized thereon (i) a first agent that provides a primary
activation signal to the T cells and (ii) a second agent which
stimulates an accessory molecule on the surface of the T cells,
[0156] wherein the multimerization reagent comprises at least one
binding site Z1 for the reversible binding of the first agent,
[0157] wherein the first agent comprises at least one binding
partner C1, wherein the binding partner C1 is able of reversibly
binding to the binding site Z1 of the multimerization reagent,
wherein the first agent is bound to the multimerization reagent via
the reversible bond formed between the binding partner C1 and the
binding site Z1, [0158] wherein the multimerization reagent
comprises at least one binding site Z2 for the reversible binding
of the second agent, [0159] wherein the second agent comprises at
least one binding partner C2, wherein the binding partner C2 is
able of reversibly binding to the binding site Z2 of the
multimerization reagent, wherein the first agent is bound to the
multimerization reagent via the reversible bond formed between the
binding partner C2 and the binding site Z2, [0160] wherein the
first agent binds to a receptor molecule on the surface of the T
cells, thereby providing a primary activation signal to the cells
and thereby activating the T cells, [0161] wherein the second agent
binds to the accessory molecule on the surface of the T cells,
thereby stimulating the activated cells, the first agent and the
second agent thereby together inducing the T cells to expand.
[0162] In this method contacting the sample that contains the
population of lymphocytes that in turn contains the T cell
population with the soluble multimerization reagent that has
immobilized thereon the first and second agent results in specific
binding of T cells to the multimerization reagent.
[0163] The contacting of the sample comprising the T cell
comprising population of lymphocytes with the multimerization
reagent can be carried out in a bioreactor such as a hollow-fiber
bioreactor (e.g. hollow fiber bioreactor of the Quantum.RTM. cell
expansion system) or a plastic bag bioreactor (e.g. Cellbag.RTM.
used in Xuri Cell Expansion System W25 from GE Healthcare).
[0164] This method further comprises contacting the population of
lymphocytes (reaction mixture containing the T cells bound to the
multimerization reagent via the first agent and the second agent)
with (i) a free first binding partner C1 or an analogue thereof
capable of disrupting the bond between the first binding partner C1
and the binding site Z1 and (ii) a free second binding partner C2
or an analogue thereof, capable of disrupting the bond between the
second binding partner C2 and the binding site Z2. By so doing the
reversible bond between said binding partner C1 of the first agent
and said binding sites Z1 as well as the reversible bond between
said binding partner C2 of the second agent and said binding site
Z2 of said multimerization reagent is disrupted, thereby releasing
in an eluate the T cells bound to the multimerization reagent via
the first agent and the second agent and stopping the expansion of
the T cells.
[0165] In this method the eluate (the reaction mixture in which the
expansion reaction has been terminated by addition of the free
first partner(s) or analogue(s) thereof) that contains the expanded
T cell population may be exposed to chromatography on a suitable
(first) stationary phase. The (first) stationary phase may be a gel
filtration matrix and/or an affinity chromatography matrix as
described in International patent application WO 2013/124474. This
gel filtration and/or affinity chromatography matrix comprises an
affinity reagent, wherein the affinity reagent comprises a binding
site Z1 and/or Z2 specifically binding to the binding partner C1
and/or C2 comprised in the first agent or the second agent. By so
doing the first agent, the second agent, the first binding partner
C1 and/or the free second binding partner C2 are immobilized on the
stationary phase. In this method, the first stationary phase is
fluidly connected to the bioreactor.
[0166] In one of the embodiments of this serial expansion the
binding sites Z1 and Z2 of the multimerization agent are identical.
In addition, a single multimerization agent may be used. When a
soluble multimerization agent is used, the T cell population (or
the expanded cell population in general) is separated from the
soluble multimerization reagent. The separation/removal might be
carried out using a second stationary phase. For this purpose, a
mixture comprising the T cells and the soluble multimerization
reagent are exposed, before or after being applied onto the first
stationary phase described above, to chromatography on a suitable
second stationary phase. This secondary stationary phase may be a
gel filtration matrix and/or affinity chromatography matrix,
wherein the gel filtration and/or affinity chromatography matrix
comprises an affinity reagent. The affinity reagent comprised on
the chromatography resin include a binding partner D that
(specifically) binds to the binding site Z1 and/or binding site Z2,
if present, of the multimerization reagent, thereby immobilizing
the multimerization reagent on the stationary phase. If a
streptavidin based multimerization agent is used and both first and
second agents have a streptavidin binding peptide as binding
partner C1 or C2, the binding partner D that is comprised in the
affinity reagent of this second stationary phase can be biotin. The
soluble oligomer of streptavidin or of a streptavidin mutein that
is used as multimerization reagent then binds to the biotin that is
usually covalently coupled to a chromatography matrix such as
biotin-sepharose.TM. that is commercially available.
[0167] In this method of serial expansion the first agent may
stimulates a TCR/CD3 complex-associated signal in the T cells and
the first agent may thus be a binding reagent that specifically
binds CD3. In addition, the accessory molecule on the T cell may be
CD28. In this case the second agent that binds the accessory
molecule is a binding reagent that specifically binds CD28.
[0168] In this method of serial expansion, the T cells may be
transfected either during or after expansion e.g. with a T cell
receptor (TCR) or a chimeric antigen receptor (CAR, also known as
artificial T cell receptor). This transfection for the introduction
of the gene of the desired receptor can be carried out with any
suitable retroviral vector, for example. The genetically modified
cell population can then be liberated from the initial stimulus
(the CD3/CD28 stimulus, for example) and subsequently be stimulated
with a second type of stimulus e.g. via the de novo introduced
receptor). This second type of stimulus may comprise an antigenic
stimulus in form of a peptide/MHC molecule, the cognate
(cross-linking) ligand of the genetically introduced receptor (e.g.
natural ligand of a CAR) or any ligand (such as an antibody) that
directly binds within the framework of the new receptor (e.g. by
recognizing constant regions within the receptor). Cf. in this
respect, Cheadle et al, "Chimeric antigen receptors for T-cell
based therapy" Methods Mol Biol. 2012; 907:645-66 or Barrett et
al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of
Medicine Vol. 65: 333-347 (2014).
[0169] In this method, the population of lymphocytes that comprises
T cells can be a population of peripheral blood mononucleated cells
(PBMC) or a population of enriched or purified T cells. The
population of lymphocytes may, for example, be derived from whole
blood, or from a non-mobilized apheresis product or a frozen tissue
preparation.
[0170] In this method of serial expansion that is based on a
soluble multimerization reagent, the first and second reagent as
well as the multimerization reagent and all other reagents and cell
populations can otherwise be used in the same manner as disclosed
above for the method that makes use of reversible between the first
or second agent and the multimerization reagent.
[0171] The invention is further directed to an arrangement of a
bioreactor and a first stationary phase for chromatography. The
bioreactor is suitable for the expansion of cells, and the
stationary phase is suitable for cell separation and removal of
reagents. The first stationary phase is a gel filtration matrix
and/or affinity chromatography matrix, wherein the gel filtration
and/or affinity chromatography matrix comprises an affinity
reagent, wherein the affinity reagent comprises a binding site Z1
specifically binding to a binding partner C1 comprised in a first
agent and/or the affinity reagent comprises a binding site Z2
specifically binding to a binding partner C2 comprised in a second
agent. The first stationary phase is thereby being suitable of
immobilizing thereon the first agent and/or the second agent, the
first binding partner C1 and/or the free second binding partner C2.
In addition the bioreactor and the stationary phase are fluidly
connected. This arrangement can be used in the serial expansion as
explained above and can be integrated into known cell expansion
systems such as the Quantum.RTM. cell expansion system) or the Xuri
Cell Expansion System W25.
[0172] In this arrangement the first stationary phase is either
comprised in a chromatography column or is a planar stationary
phase. The arrangement may further comprises a second stationary
phase which is fluidly connected to the first stationary phase. The
secondary stationary phase may be a gel filtration matrix and/or
affinity chromatography matrix, wherein the gel filtration and/or
affinity chromatography matrix comprises an affinity reagent. This
affinity reagent may comprise a binding partner D that
(specifically) binds to the binding site Z1 of the multimerization
reagent, thereby being suitable of immobilizing the multimerization
reagent on the stationary phase.
[0173] The invention is further directed to an apparatus for
purification and expansion of a population of cells, the apparatus
comprising at least one arrangement of a bioreactor and a first
stationary phase or a second stationary phase for chromatography as
defined above.
[0174] The apparatus may further comprise a plurality of
arrangements of a bioreactor and a stationary phase being fluidly
connected in series.
[0175] The apparatus may comprise a sample inlet being fluidly
connected to the bioreactor of the arrangement of a bioreactor and
the stationary phase for chromatography. The apparatus may also
comprise a sample outlet for purified and expanded target cells,
the sample outlet being fluidly connected to the stationary phase
of the last of the at least one arrangement of a bioreactor and the
stationary phase for chromatography.
[0176] Finally, the apparatus may be designed as a functionally
closed system.
[0177] As one of ordinary skill in the art will readily appreciate
from the disclosure of the present invention, other compositions of
matter, means, uses, methods, or steps, presently existing or later
to be developed that perform substantially the same function or
achieve substantially the same result as the corresponding
exemplary embodiments described herein may likewise be utilized
according to the present invention.
EXPERIMENTAL EXAMPLES
Example 1: Stimulation/Expansion of CD3+ T Responder Cells with
.alpha.CD3/.alpha.CD28 Fab Fragments that Were Reversibly
Immobilized on Beads Coated with the Streptavidin Mutein
Strep-Tactin.RTM..
[0178] 300.000 CD3+CD62L-responder T cells (Tresp, isolated by
serial magnetic enrichment from a non-mobilized donor apheresis
product) were labeled with 3 .mu.M CFSE and stimulated with 5 .mu.l
of a 15 .mu.l preparation of Streptactin.RTM. beads (10 mg magnetic
particles/ml, loaded with 35 .mu.g Streptactin.RTM./mg beads)
either loaded with 0.5 .mu.g .alpha.CD3 Fab fragment alone, 0.5
.mu.g .alpha.CD28 Fab fragment alone or a mixture of 0.5 .mu.g
.alpha.CD3 Fab fragment and 0.5 .mu.g .alpha.CD28 Fab.
[0179] The .alpha.CD3 Fab fragment used was derived from the CD3
binding monoclonal antibody produced by the hybridoma cell line
OKT3. The hybridoma cell line OKT3 and the OKT3 antibody are
described in U.S. Pat. No. 4,361,549, the cell line has been
deposited under accession number ATCC.RTM. CRL-8001.TM.). The CD28
Fab used was derived from the monoclonal anti-human CD28 antibody
CD28.3 (Vanhove et al, BLOOD, 15 Jul. 2003, Vol. 102, No. 2, pages
564-570). The nucleotide sequence of the variable domains of this
antibody CD28.3 has been deposited in GenBank in the form of a
synthetic single chain Fv construct anti-human CD28 antibody
scFv28.3 under GenBank accession number AF451974.1).
[0180] Both Fab fragments were recombinantly produced in E. coli as
described in International patent applications WO2013/011011 and WO
2013/124474 carrying as constant domains (CH1 and Ckappa) an IgG1
consensus sequence. The heavy chain of both Fab fragments was
carboxy-terminally fused with a sequential arrangement of two
streptavidin binding modules (SAWSHPQFEK(GGGS).sub.2GGSAWSHPQFEK,
(SEQ ID NO: 07)), that is commercially available as
"Twin-Strep-tag.RTM. from IBA GmbH, Gottingen, Germany) The
.alpha.CD3 Fab fragment was used as first agent with the
streptavidin binding peptide serving as binding partner C 1 and the
.alpha.CD28 Fab fragment was used as second agent with the
streptavidin binding peptide serving as binding partner C2. The
(tetrameric) streptavidin mutein "Strep-tactin.RTM. serves as
multimerization reagent on which both Fab fragments were reversibly
immobilized.
[0181] In the expansion experiment, Tresp cells stimulated with
blank beads (no Fab) served as negative control. Tresp cells were
seeded in triplets in 48-well plates along with 300.000 CD3 cells
autologous feeder cells (irradiated with 30Gy) in 3 ml complete
cell culture medium (RPMI (Gibco) supplemented with 10% (v/v) fetal
calf serum, L-glutamine, b-mercapto ethanol, HEPES, penicillin,
streptomycine and gentamycine) supplemented with 10 U/ml
interleukin 2 (IL-2). The cells were incubated at 37.degree. C.
without media exchange and analyzed after 4 days by FACS analysis.
FACS staining and analysis was done after 10 min incubation with
100 .mu.M D-biotin. One representative plot for each condition is
shown in FIG. 4. Plots show live CD3+ cells that were stained with
propidium iodide (PI) for live/dead discrimination. FIG. 4a is a
histogram showing size-distribution (forward scatter) of stimulated
cells. FIG. 4a shows that a specific cell population of Tresp cells
was stimulated and expanded (increase in size/number compared to
the unstimulated "beads only" control) when incubated in the
presence of beads on which a mixture of 0.5 .mu.g .alpha.CD3 Fab
fragment and 0.5 .mu.g .alpha.CD28 Fab was immobilized, after being
stimulated in vitro with .alpha.CD3/.alpha.CD28 Fab fragments that
were reversibly immobilized on beads coated with the streptavidin
mutein Strep-tactin.RTM.. FIG. 4B depicts histograms of the
dilution of the proliferation dye CFSE representing the degree of
proliferation according to the number of cells per cell division
(indicated on top of FIG. 4B, 0 represents undivided cells; 5
represents cells that have gone through at least 5 divisions). It
can be seen from FIG. 4B that the population of T cells stimulated
with the beads on which a mixture of 0.5 .mu.g .alpha.CD3 Fab
fragment and 0.5 .mu.g .alpha.CD28 Fab was immobilized have mostly
gone through three cell divisions and represent a more uniform
proliferation pattern than with a single stimulus alone (small
number of cells within the undivided peak "0"). The increased
absolute amount of proliferation (more cells have proliferated
uniformly after 4 d stimulation with .alpha.CD3 and .alpha.CD28
functionalized beads) is also represented by a more intense
consumption of media as depicted by a indicator color change to
yellow in FIG. 4C.
Example 2: Analysis of the Differential Intracellular Calcium
Mobilization in Jurkat Cells
[0182] Real-time low-cytometric analysis of the differential
intracellular calcium mobilization induced in Jurkat cells that are
either labeled with the .alpha.CD3 antibody clone OKT3 or with Fab
fragments of OKT3 being multimerized with Strep-tactin.RTM. was
examined here.
[0183] For this purpose, Jurkat cells were loaded with the
calcium-sensitive dye Indo-1-AM and calcium release was triggered
by injection of either .alpha.CD3 monoclonal antibody OKT3
(produced by the hybridoma cell line OKT3, see above, black
squares) or .alpha.CD3 Fab fragments (derived from the parental
cell line OKT3) that were multimerized by reversible binding of its
streptavidin binding peptide to soluble Strep-Tactin fluorescently
conjugated with phycoerythrin. In the case of the intact multimeric
OKT3 Fab-Strep-Tactin complexes, the calcium release was triggered
over an identical time period as with the parental antibody clone
(dark grey triangles). Activation of cells could be completely
avoided by injection of D-biotin treated, pre-dissociated
Fab-Strep-Tactin complexes (light grey circles) identical to
injection of the PBS negative control (inverted white triangles).
Application of ionomycine served as positive control for calcium
influx. Time-resolved changes in intracellular Ca.sup.2-
concentration were monitored by flow-cytometry based on the change
in FL6/FL7 ratio. It can be seen from FIG. 5A that both the
parental antibody OKT3 as well as the multimerized monovalent Fab
fragment of OKT3 effected calcium release, meaning that the
multimerized monovalent Fab fragment of OKT3 is essentially as
functional as the parental antibody. Notably, the multimeric OKT3
Fab fragment was not able to trigger calcium release if biotin was
added to Strep-tactin on which the OKT3 Fab fragment was
immobilized prior to the addition of the Streptactin-OKT3 Fab
fragment. In this case, the biotin disrupted the reversible bond
formed between Strep-tactin as multimerization agent and the OKT3
Fab fragment. The monovalent Fab fragment was therefore displaced
from the multimerisation agent and after dissociation was not able
to trigger calcium release by binding to CD3 of the Jurkat
cells.
[0184] In the experiments shown in FIG. 5B indo-1-AM-labeled Jurkat
cells were activated by OKT3 derived .alpha.CD3
Fab-Strep-Tactin-complexes as described in FIG. 5a. Injection of
intact (upper graph) or pre-dissociated complexes (lower graph)
served as positive or negative controls respectively. In addition,
stimulation of cells with intact Fab-Strep Tactin-complexes
followed by subsequent injection of D-biotin (near the peak
activation at t=1405) resulted in abrupt disruption of .alpha.CD3
Fab-multimer signaling (middle graph). Injection of ionomycine into
the pre-dissociated Fab complex group served as positive control.
Data are representative of three different experiments.
Importantly, FIG. 5B shows that the addition of D-biotin to the
sample rapidly displaces the Fab fragment from the Strep-tactin
multimerization agent, thereby effectively terminating the calcium
release even under ongoing calcium stimulation and demonstrating
that the dissociated OKT3 Fab fragment is not any longer
biologically active. Likewise, the multimeric OKT3 Fab fragment was
also not able to trigger calcium release when biotin was added to
the Strep-tactin-OKT3 Fab fragment multimer prior to the addition
of the Streptactin-OKT3 Fab sample to the Jurkat cells.
Example 3: Reversible Staining of Cells by CD3 Fab-Multimers
[0185] This Example examines the reversible staining of cells by
CD3 Fab-multimers. Freshly isolated PBMCs were stained with either
the .alpha.CD3 monoclonal antibody clone OKT3 (left dot plot,
parental clone for the Fab-multimers) or cognate phycoerythrine
(PE)-labeled OKT3 Fab-multimers and analyzed either before (second
left column) or after treatment with D-biotin (middle column).
Remaining Fab monomers were then detected after subsequent washing
steps using fresh PE-labeled Strep-Tactin.RTM. (second right
column) Secondary Fab-multimer staining of reversibly stained cells
served as control (right column) Only live CD3 cells which are
negative in staining with propidium iodide (PI) for live/dead
discrimination are shown in FIG. 6. Numbers in dot plots indicate
the percentage of cells within gates. This experiment shows that
the staining of CD3+ PBMCs with an anti-CD3 Fab fragment
multimerized with Streptactin as multerization reagent is fully
reversible by addition of D-biotin and that the monovalent Fab
fragment alone does not bind to the CD3 molecule present on
PBMCs.
Example 4: Reversible Isolation of Cells by CD28 Fab-Multimers
[0186] This Example shows the isolation of cells by reversible
binding of anti-CD28 Fab fragments multimerized with
Strep-Tactin.RTM. magnetic particles (the magnetic particles are
available from IBA GmbH Gottingen, Germany). The Fab fragments
derived from the antibody CD28.3 described in Example 1 above were
used for this purpose. CD28+ cells were selected/isolation by
Fab-multimer magnetic cell selection from freshly isolated PMBCs as
essentially described in International Patent Application
WO2013/011011. Before selection cells were control stained with
either the cognate fluorescent .alpha.CD28-multimers (left dot
plot) or with an antibody directed against the immunoglobulin kappa
light chain (second left dot plot, .alpha.-Ig kappa mAb) as a
control staining. After selection, CD28+ cells were treated with
D-biotin and subsequently washed to remove magnetic beads and
Fab-monomers. Liberated CD28+ cells were subsequently (re-) stained
either with CD28 Fab-multimers (second right dot plot) or with the
.alpha.-Igkappa mAb (right dot plot) to detect potentially
remaining Fab-monomers. Only live (PI.sup.negative) CD3+ cells are
shown. Numbers in dot plots indicate the percentage of cells within
gates. FIG. 7 shows that CD28+ cells can be isolated from PMBC
using such multimerized anti-CD28 Fab fragment and that all
isolation reagents including the anti CD28 Fab-monomers can be
removed after selection.
Example 5: Stimulation/Expansion of CD3+ T Responder Cells with
.alpha.CD3/.alpha.CD28 Fab Fragments that Were Reversibly
Immobilized on Soluble Strep-Tactin
[0187] In this example CD3+ T responder cells (isolated by magnetic
selection from a sample of fresh PBMCs obtained from a Ficoll
gradient) were expanded after in vitro stimulation with
.alpha.CD3/.alpha.CD28 Fab fragments that were reversibly
immobilized on soluble oligomeric Strep-tactin.RTM. acting as a
soluble multimerization reagent. The oligomeric Strep-tactin.RTM.
was obtained by polymerizing Strep-tactin.RTM. with sulfo SMCC
(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
product #22122 Thermo Scientific) and iminothiolan (product #26101
Thermo Scientific) according to the protocol of the manufacturer
(Thermo Scientific). The oligomeric streptavidin were separated
from monomeric (unreacted) and dimeric streptavidin mutein by size
exclusion chromatography and the so obtained fraction of the
oligomeric streptavidin mutein (n.gtoreq.3) was used as soluble
multimerization reagent.
[0188] For the in vitro expansion, 300.000 CD3+ responder T cells
(Tresp) were labeled with 2 .mu.M Carboxyfluorescein succinimidyl
ester (CFSE) and stimulated with varying amounts of a preparation
of soluble Strep-tactin.RTM. oligomers on which a combination of
the above described .alpha.CD3 OKT3 Fab fragment and the
.alpha.CD28 Fab fragment of the antibody 28.3 (both carrying the
above-mentioned Twin-Strep-tag.RTM. as streptavidin binding peptide
at the heavy chain) were immobilized. ("1.times." corresponds to 3
.mu.g multimerized Streptactin functionalized with 0.5 .mu.g of the
.alpha.CD3- and 0.5 .mu.g .alpha.CD28 monomeric Fab fragment, the
numbers "0.5x", "2x" and "5.times." indicate the respective n-fold
amount of "1.times."). Tresp cells either left unstimulated or were
stimulated with blank Strep-tactin multimers (no Fab) served as
negative controls. Tresp cells were seeded in duplicates in 48-well
plates along with 300.000 CD3 negative autologous feeder cells
(irradiated with 30Gy) in 1 ml cell culture medium supplemented
with 20 U/ml IL-2. Cells were incubated at 37.degree. C. without
media exchange and proliferation was analyzed according to CFSE
dilution after 5 days by FACS analysis. FIG. 8A shows the increase
in the size distribution of proliferating cells after 5 days in
culture compared to the negative controls. FIG. 8B shows that CD3+
Tresp cells were properly stimulated and proliferated vigorously
when incubated with soluble oligomeric Strep-tactin.RTM. (as
compared to solid Streptactin magnetic particles in FIG. 4) on
which a mixture of .alpha.CD3 Fab and .alpha.CD28 Fab fragments
were immobilized. The results in FIGS. 8a and 8b indicate that
under these in vitro conditions most of the CD3+ T responder cells
divided (2 to 5 cell divisions) after engagement of the surface
CD28 and TCR/CD3 complex with the .alpha.CD3 and .alpha.CD28 Fab
fragments that were reversibly immobilized on soluble
Strep-tactin.RTM. oligomers. After in vitro expansion the soluble
Fab-Strep-Tactin stimulation reagents were dissociated and removed
after D-biotin treatment. The dissociation and removal of monomeric
Fab fragments was flow-cytometrically analyzed by restaining cells
with phycoerythrine label Strep-Tactin.RTM.) (ST-PE). A
representative histogram (dark grey histogram) is shown compared to
the appropriate ST-PE only negative control (light gray histogram).
It can be seen from FIG. 8C that both Fab fragments had completely
dissociated and were entirely removed from the expanded cells. FIG.
8D shows the absolute number of live (trypan blue negative) cells
after 5 days. The number was counted using a Neubauer counting
chamber and plotted against the respective stimulation condition.
Median cell numbers are shown in FIG. 8D; error bars indicate
standard deviation (SD). FIG. 8D shows that all which mixtures of
.alpha.CD3 Fab fragments and .alpha.CD28 Fab fragments that were
immobilized on a soluble Strep-tactin multimerization reagent were
equally effective in expanding the CD3+ cells and resulted in an
approx. 4-fold increase of absolute cell numbers.
Example 6: Kinetics of Proliferation of Purified CD4+ and CD8+ T
Responder Cells Stimulated In Vitro with Reversible
.alpha.CD3/.alpha.CD28 Fab-Streptamer Multimers Without Medium
Exchange
[0189] In this example the expansion kinetics of proliferation of
purified CD4+ and CD8+ T responder cells (Tresp) that were
stimulated in vitro with .alpha.CD3/.alpha.CD28 Fab fragments that
were reversibly immobilized soluble oligomeric streptavidin muteins
were examined For this purpose, soluble oligomeric
Strep-tactin.RTM. mutein of two different sizes served as soluble
multimerization reagent. The first kind of oligomeric
Strep-tactin.RTM. was the fraction of the oligomeric streptavidin
mutein (n.gtoreq.3) obtained in Example 5 (also referred herein as
"conventional Streptactin.RTM. backbone", illustrated by the
triangle symbol with the tip on top in FIG. 13). The second kind of
this oligomeric streptavidin mutein used as soluble multimerization
reagent was an oligomeric streptavidin mutein (n.gtoreq.3) that was
reacted with biotinylated human serum albumin (also referred herein
as "large Streptactin.RTM. backbone).
[0190] In this example 500.000 purified CD4+ or CD8+ responder T
cells (Tresp) were separately stimulated with these two different
Streptamer multimers as explained above, i.e. with either the
Streptactin backbone of Example 5 (using a solution with a
concentration of 1 mg oligomeric streptavidin mutein/ml)) or with
the large Streptactin backbones (0.1 mg/ml). 3 .mu.l of the both
different backbones were either loaded with a combination of 0.5
.mu.g of the .alpha.CD3 and 0.5 .mu.g .alpha.CD28 Fab used in the
earlier Examples that carried a streptavidin binding peptide
SAWSHPQFEK(GGGS).sub.2GGSAWSHPQFEK (SEQ ID NO: 07) at the
C-terminus of the heavy chain of the Fab fragment. In addition, 4.5
.mu.l of the conventional Streptactin backbone was loaded with 0.5
.mu.g .alpha.CD3 Fab fragment, 0.5 .mu.g .alpha.CD8 Fab fragment
(IBA GmbH Gottingen, that also carries at the C-terminus of the Fab
fragment the streptavidin binding peptide
SAWSHPQFEK(GGGS).sub.2GGSAWSHPQFEK (SEQ ID NO: 07) and 0.5 .mu.g
.alpha.CD28 Fab fragment. Untreated (unstimulated) Tresp cells
served as negative control and Tresp cells stimulated with
commercially available Dynabeads (beads on which .alpha.CD3 and
.alpha.CD28 monoclonal antibodies are irreversible immobilized) as
positive control. Tresp cells were seeded in duplicates in 48-well
plates in lml cell culture medium (RPMI 1640 (Gibco) supplemented
with 10% (v/v fetal calf serum, 0.025% (w/v) L-glutamine, 0.025%
(w/v) L-arginine, 0.1% (w/v) HEPES, 0.001% (w/v) gentamycine,
0.002% (w/v) streptomycine, 0.002% (w/v) peniciline) supplemented
with 30 U/ml IL-2. Cells were incubated at 37.degree. C. without
media exchange and cell count was analyzed after 1, 3 and 6 days.
In the experiments of FIG. 13 the expansion was carried out without
medium exchange. The results for the CD4+ T responder cells are
shown in FIG. 13A, the results for the CD8+ T responder cells are
shown in FIG. 13B, with the graphs representing degree of
proliferation according to the number of cells harvested per time
point for CD4+ Tresp (FIG. 13A) and for CD8+ Tresp in FIG. 13B.
[0191] As can be seen from FIG. 13A the "smaller" soluble
multimerization reagent on which .alpha.CD3 and .alpha.CD28 Fab
fragments were reversibly immobilized provided for the same amount
of expansion of CD4+ T cells as Dynabeads (which are so far the
standard reagent for the expansion of T cells), while the "larger"
oligomeric soluble streptactin provided for even better expansion
compared to Dynabead. This improvement might be caused by the
soluble "larger oligomeric multimerization reagent" being able to
bind to more T cells at the same time than the "smaller" soluble
oligomer, thereby being able to stimulate more CD4+ T cells than
the "smaller" oligomer.
[0192] As evident from FIG. 13B, using the soluble multimerization
reagents of the present invention CD8+ T cells could be expanded
within the first 3 days at least as efficiently as with Dynabeads.
Notably, in this time period, the expansion experiment that used a
soluble multimerization reagent that in addition to .alpha.CD3 and
.alpha.CD28 Fab fragments (as first and second agent) carried
reversibly immobilized thereon .alpha.CD8 Fab fragment, showed the
best degree of expansion under these culturing conditions. This
indicates that it is possible by using a stimulus that is specific
for a particular sub-population of cells (here the .alpha.CD8 Fab
fragment) to increase or modulate the selectivity of the expansion,
thereby being able to obtain larger amounts of a desired cell
(sub)-population.
[0193] Thus, summarizing the above, Example 6 shows that the
functionality of the soluble multimerization reagent used in the
present invention in terms of triggering expansion of T cells is
comparable to the current standard methodology of using Dynabeads
for this purpose. However, since the stimulation can be controlled
(and terminated, if wanted) by adding a competitor such as biotin
in the case of a streptavidin based reversible interaction between
the first and second agent and the multimerization reagent, the
present invention provides a significant advantage over the
Dynabeads technology since the expansion conditions can be
optimized (it would for example be possible to stop the stimulation
in the experiment of FIG. 13B after 3 days). In addition, since the
soluble multimerization reagent can be easily removed from the
reaction (for example, by immobilizing the reagent on a
biotinylated column after the expansion reaction), the expansion
method of the invention can be carried out and automated in closed
systems that are, for example, needed for GMP production of cells
for therapeutic purposes, without having to deal with the removal
of beads such as Dynabeads.
Example 7: Kinetics of Proliferation of Purified CD4+ and CD8+ T
Responder Cells Stimulated In Vitro with Reversible
.alpha.CD3/.alpha.CD28 Fab-Streptamer Multimers with Medium
Exchange
[0194] Also in this example the expansion kinetics of proliferation
of purified CD4+ and CD8+ T responder cells (Tresp) that were
stimulated in vitro with .alpha.CD3/.alpha.CD28 Fab fragments that
were reversibly immobilized on soluble oligomeric streptavidin
muteins were examined For this purpose, soluble oligomeric
Strep-tactin.RTM. mutein of two different sizes served as soluble
multimerization reagent. The first kind of oligomeric
Strep-tactin.RTM. was the fraction of the oligomeric streptavidin
mutein (n.gtoreq.3) obtained in Example 5 (also referred herein as
"conventional Streptactin.RTM. backbone", illustrated by the
triangle symbol with the tip down in FIG. 13). The second kind of
this oligomeric streptavidin mutein used as soluble multimerization
reagent was obtained by reacting the oligomeric Strep-tactin
(n.gtoreq.3) obtained in Example 5 with biotinylated human serum
albumin This soluble oligomeric multimerization reagent is also
referred herein as "large Streptactin.RTM. backbone.
[0195] In this example, 400.000 purified CD4+ or CD8+ responder T
cells (Tresp) were separately stimulated with these two different
Streptamer multimers as explained above, i.e. with either the
Streptactin backbone of Example 5 (1.0 mg/ml) or with the large
Streptactin backbones (0.1 mg/ml). 3 .mu.l of both the different
backbones were either loaded with a combination of 0.5 .mu.g
.alpha.CD3 and 0.5 .mu.g .alpha.CD28 Fab fragments described above.
In addition, 4.5 .mu.l of the Streptactin backbone of Example 5 was
loaded with 0.5 .mu.g .alpha.CD3, 0.5 .mu.g .alpha.CD8 Fab and 0.5
.mu.g .alpha.CD28 Fab fragment as described above. Untreated
(unstimulated) Tresp cells served as negative control and Tresp
cells stimulated with Dynabeads (on which .alpha.CD3 and
.alpha.CD28 monoclonal antibodies are irreversible immobilized) as
positive control. Tresp cells were seeded in duplicates in 48-well
plates in lml cell culture medium supplemented with 30 U/ml IL-2.
Cells were incubated at 37.degree. C. with media exchange on day 3
and cell count was analyzed after 1, 3 and 6 days. The results for
the CD4+ T responder cells are shown in FIG. 14A, the results for
the CD8+ T responder cells are shown in FIG. 14B, with the graphs
representing degree of proliferation according to the number of
cells harvested per time point for CD4+ Tresp (FIG. 14A) and for
CD8+ Tresp in FIG. 14B.
[0196] As can be seen from FIG. 14A the soluble multimerization
reagents of the present invention on which .alpha.CD3 and
.alpha.CD28 Fab fragments were reversibly immobilized provided for
better expansion of CD4+ T cells than Dynabeads.
[0197] As evident from FIG. 14B, using the soluble multimerization
reagents of the present invention CD8+ T cells could be expanded
within the first 6 days at least as efficiently as with Dynabeads.
Notably, in this time period, the expansion experiment that used
the larger soluble multimerization reagent that carried .alpha.CD3
and .alpha.CD28 Fab fragments (as first and second agent) showed
the best degree of expansion under these culturing conditions. This
might again be caused by the soluble "larger oligomeric
multimerization reagent" being able to bind to more T cells at the
same time than the "smaller" soluble oligomer, thereby being able
to stimulate more CD4+ T cells than the "smaller" oligomer.
Example 8: Expansion Kinetics of Purified CD4+ and CD8+ T Cell
Cultures with or Without Medium Exchange
[0198] In this Example the combined data from Examples 6 and 7 were
normalized on input cell number for the "smaller" soluble
multimerization reagent and positive and negative control. No
normalization data was obtained on the "larger" multimerization
reagent. As explained in Examples 6 and 7, 400.000 to 500.000 CD4+
or CD8+ responder T cells (Tresp) were stimulated with 3 .mu.l of a
preparation of Streptactin multimers (1 mg/ml; on which 0.5 .mu.g
.alpha.CD3 Fab fragment and 0.5 .mu.g .alpha.CD28 Fab fragment were
immobilized. Untreated (unstimulated) Tresp cells served as
negative control and Tresp cells stimulated with Dynabeads as
positive control. Tresp cells were seeded in duplicates in 48-well
plates in lml cell culture medium supplemented with 30 U/ml IL-2.
Tresp cells were seeded in duplicates in 48-well plates in 1 ml
cell culture medium supplemented with 30 U/ml IL-2. Cells were
incubated at 37.degree. C. with media exchange (straight lines in
FIG. 15) or without media exchange (dashed lines in FIG. 15) on day
3 and cell count was analyzed after 1, 3 and 6 days. As evident
from the normalized data of FIG. 15A, the "smaller" soluble
multimerization reagent on which .alpha.CD3 and .alpha.CD28 Fab
fragments were reversibly immobilized yielded an about 2.5 fold
expansion of CD4+ T cells, while the expansion using Dynabeads
yielded an about 1.8 fold expansion rate. Thus, the use of a
soluble multimerization reagent of the invention even provides for
an improvement in the expansion of CD4+ T cells over Dynabeads.
Similarly, FIG. 15B, confirms that using the soluble
multimerization reagents of the present invention CD8+ T cells
could be expanded within the first 3 days at least as efficiently
as with Dynabeads.
Example 9: Early Cluster Formation After Activation of Purified
CD4+ and CD8+ T Responder Cells Stimulated In Vitro with Reversible
.alpha.CD3/.alpha.CD28 Fab-Streptamer Multimers
[0199] In this Example, 400.000 CD4+ or CD8+ responder T cells
(Tresp) were stimulated with 3 .mu.l of a preparation of oligomeric
Streptactin multimerization reagent (1 mg/ml) loaded with a
combination of 0.5 .mu.g .alpha.CD3- and 0.5 .mu.g .alpha.CD28 Fab.
Untreated (unstimulated) Tresp cells served as negative control and
Tresp cells stimulated with Dynabeads as positive control. Tresp
cells were seeded in duplicates in 48-well plates in lml cell
culture medium supplemented with 30 U/ml IL-2. Cells were incubated
at 37.degree. C. and microscopically analyzed after 1 and 2 days.
Stimulation of CD4+ Tresp (FIG. 16A) and CD8+ Tresp (FIG. 16B) are
shown for Dynabeads (middle row) and Streptamer multimers (lower
row) respectively. The photographs represent degree of cluster
formation: For better visibility exemplary clusters are indicated
by circles for the stimulation with soluble streptavidin mutein
oligomers in FIG. 16A and FIG. 16B. Clusters within the Dynabead
stimulation are readily visibly by accumulation of dark stimulatory
particles. As evident, both for CD4+ and CD8+ T cells early
clusters formed when using the expansion method of the invention
that employs a soluble oligomeric multimerization reagent.
Example 10: Expansion Kinetics & Phenotype of Polyclonal
Activated/Expanded Bulk CD3+ Central Memory T Cells (Tcm)
[0200] In this Example, 500.000 CD3+CD62L+CD45RA- responder Tcm
cells (Tresp) were stimulated with 3 .mu.l of a preparation of the
soluble oligomeric Streptactin of Example 5 (1 mg/ml) that was
either loaded with a combination of 0.5 .mu.g .alpha.CD3 and 0.5
.mu.g .alpha.CD28 Fab. Furthermore, 4.5 .mu.l of a preparation of
Streptactin multimers loaded with 0.5 .mu.g .alpha.CD3, 0.5 .mu.g
.alpha.CD8 Fab and 0.5 .mu.g .alpha.CD28 Fab was used as an
additional stimulation condition. Untreated (unstimulated) Tresp
cells served as negative control and Tresp cells stimulated with
Dynabeads (on which .alpha.CD3 and .alpha.CD28 monoclonal
antibodies are irreversible immobilized) as positive control. Tresp
cells were seeded in 48-well plates in lml cell culture medium
supplemented with 30 U/ml IL-2 only or 30 U/ml IL-2 and 5ng/ml
IL-15. Cells were incubated at 37.degree. C. with media exchange
every 3 days and cell count was analyzed after 7 and 14 days.
Graphs represent degree of proliferation according to the number of
cells harvested per time point, in FIG. 17A only IL-2 supplemented
media and in FIG. 17B IL-2 and IL-15 supplemented media. As can be
seen from both FIG. 17A and FIG. 17B, the soluble multimerization
reagent that has reversibly bound thereon CD3 Fab fragment and
.alpha.CD28 Fab fragment yields better cell expansion than the
Dynabeads. As further shown by the flow-cytometric analysis of
CD62L and CD127 surface expression after 14 days of culture in
variable cytokine milieus of FIG. 17C, the experimental approaches
using soluble multimerization reagents of the present invention
retain, under both conditions chosen here, a higher content of
CD127-expressing long-lived memory T cells than expansion with
Dynabeads. This illustrates a further advantage of the methods of
the present invention.
Example 11: Selective Antigen-Specific Expansion of Tcm Responder
Cells Out of Bulk CD3+ Central Memory T Cells (Kinetics &
Phenotype)
[0201] In this Example, the kinetics and the phenotype of selective
Antigen specific (Ag-specific) expansion out of purified
CD3+CD62L+CD45RA- Tcm responder cells was examined
[0202] In more detail, CD3+CD62L+CD45RA- Tcm responder cells were
stimulated in vitro with both a peptide:MHC molecule complex (that
acts as first agent that provides a primary activation signal to
the cells) and an .alpha.CD28 Fab fragment (that acts as second
reagent that stimulates an accessory molecule on the surface of the
cells). Both the complex of antigen specific peptide with the MHC
molecule and the .alpha.CD28 Fab fragment were reversibly
immobilized on the soluble oligomeric streptavidin mutein (with
n.gtoreq.3) described in Example 5. The peptide that was used for
the antigen specific expansion was the peptide CRVLCCYVL (SEQ ID
NO: 06), amino acids 309-317 of the immediate-early 1 protein
(described in Ameres et al, PLOS Pathogens, May 2013, vol. 9, issue
5, e1003383) representing an HLA-C7/IE-1 epitope that is specific
for cytomegalovirus (CMV). The MHC I molecule that presents the
peptide carries at the C-terminus of the a chain (heavy chain) the
streptavidin binding peptide (SAWSHPQFEK(GGGS).sub.2GGSAWSHPQFEK,
(SEQ ID NO: 07) that is commercially available as
"Twin-Strep-tag.RTM." from IBA GmbH, Gottingen, Germany)
[0203] For this purpose, 500.000 CD3+CD62L+CD45RA- responder Tcm
cells (Tresp) were stimulated Ag-specifically using 3 .mu.l of a
preparation of soluble oligomeric Streptactin multimerization
reagent functionalized with 0.5 .mu.g of the peptide:MHC class I
complexes equipped with the streptavidin binding peptide and with
0.5 .mu.g of the .alpha.CD28 Fab described above. As an
alternative, 4.5 .mu.l of a of preparation of the Streptactin
multimerization reagent were loaded with 0.5 .mu.g of these
peptide:MHC class I complexes, 0.5 .mu.g CD8 aFab and 0.5 .mu.g
.alpha.CD28 Fab. For comparison, polyclonal stimulation was
performed, using 3 .mu.l of a preparation of Streptactin
multimerization reagent (1 mg/ml) either loaded with a combination
of 0.5 .mu.g .alpha.CD3 Fab and 0.5 .mu.g .alpha.CD28 Fab. Again as
the alternative stimulation condition described above, 4.5 .mu.l of
a preparation of Streptactin multimerization reagent reversibly
loaded with 0.5 .mu.g .alpha.CD3 Fab, 0.5 .mu.g .alpha.CD8 Fab and
0.5 .mu.g .alpha.CD28 Fab was used. Untreated (unstimulated) Tresp
cells served as negative control and Tresp cells stimulated
polyclonal with Dynabeads (beads on which .alpha.CD3 and
.alpha.CD28 monoclonal antibodies are irreversible immobilized) as
positive control. Tresp cells were seeded in 48-well plates in lml
cell culture medium supplemented with 30 U/ml IL-2 and 5ng/ml
IL-15. Cells were incubated at 37.degree. C. with media exchange
every 3 days and cell count was analyzed after 7 and 14 days. The
exemplary flow-cytometric analysis for the fraction of Ag-specific
cells that was stimulated/expanded via the soluble strept-tactin
oligomer on which the peptide:MHC-I complex for an HLA-C7/IE-1
epitope (for CMV) was immobilized (FIG. 18A) show that these
antigen-specific T cells were specifically expanded. The graphs of
FIG. 18B to FIG. 18E (that represent the degree of expansion of
distinct Ag-specificities according to the number of peptide:MHCI
multimer-positive cells harvested per time point in analogy to the
expansion experiment shown in FIG. 18A) show that, the
multerimerization reagent that uses the respective complex of the
Ag-specific peptide and MHC 1 molecule provided for the highest
number of expanded cells (ranging from an twentyfold increase in
the number of cells for the Ag-specific cells that recognize the
pp65 epitope of CMV (amino acids 341-350 (QYDPVAALF, (SEQ ID NO:
08)) restricted by HLA-A2402) (see FIG. 18B) to an 98 fold increase
in the number of Ag-specific cells that recognize the
HLA-B7/IE-1.sub.309-317 epitope (CRVLCCYVL (SEQ ID NO: 06)) of CMV
(see FIG. 18E), thereby showing that the expansion method of the
present invention is fully applicable to the expansion of
Ag-specific cells. Finally, the exemplary flow-cytometric analysis
of CD62L and CD127 surface expression after 14 days of culture for
HLA-B7/Hexon5 epitope (for adenovirus) shown in FIG. 18F further
confirms that experimental approaches using the soluble
multimerization reagents of the present invention retain a higher
content of CD127-expressing long-lived memory T cells in polyclonal
and Ag-specific stimulatory conditions.
Example 12: Selective Ag-Specific Expansion Kinetics &
Phenotype of Bulk Central Memory T Cells
[0204] This Example examines the kinetics of selective Ag-specific
expansion out of purified CD3+CD62L+CD45RA-Tcm responder cells that
were stimulated in vitro with a) antigen specific peptide MHC I
complexes and b) .alpha.CD28 Fab fragments that were reversibly
immobilized as first and second agent on soluble oligomeric
streptavidin muteins.
[0205] For this purpose 500.000 CD3+CD62L+CD45RA- responder Tcm
cells (Tresp) were stimulated Ag-specifically using 3 .mu.l of a
preparation of Streptactin multimerization reagent functionalized
with 0.5 .mu.g peptide:MHC class I complexes equipped with a
streptavidin binding peptide (the specific peptide represents amino
acids 114-124 (CPYSGTAYNSL, SEQ ID NO: 10) of the Hexon 5 protein
of adenovirus) restricted by HLA-B07) and 0.5 .mu.g .alpha.CD28
Fab. As an alternative, 4.5 .mu.l of a preparation of Streptactin
multimerization reagent loaded with 0.5 .mu.g this peptide:MHC
class I complex, 0.5 .mu.g .alpha.CD8 Fab and 0.5 .mu.g .alpha.CD28
Fab. For comparison, polyclonal stimulation was performed, using 3
.mu.l of a preparation of Streptactin multimerization reagent (1
mg/ml) either loaded with a combination of 0.5 .mu.g .alpha.CD3 Fab
and 0.5 .mu.g .alpha.CD28 Fab. Again as the alternative stimulation
condition described above, 4.5 .mu.l of a preparation of
Streptactin multimers loaded with 0.5 .mu.g .alpha.CD3 Fab, 0.5
.mu.g .alpha.CD8 Fab and 0.5 .mu.g .alpha.CD28 Fab was used.
Untreated (unstimulated) Tresp cells served as negative control and
Tresp cells stimulated polyclonal with Dynabeads as positive
control. Tresp cells were seeded in 48-well plates in lml cell
culture medium supplemented with 30 U/ml IL-2 and 5 ng/ml IL-15.
Cells were incubated at 37.degree. C. with media exchange every 3
days and cell count was analyzed after 7 and 14 days. The pictures
shown in FIG. 19 represent degree of cluster formation on day 5,
exemplary Ag-specific stimulation is illustrated for the
HLA-B7/Hexon 5 epitope of adenovirus. As can be seen from FIG. 19,
such adenovirus antigen specific cells could be specifically
expanded from the original CD3+CD62L+CD45RA-Tcm responder
population.
Example 13: Yield and Phenotype of Expanded CD8+ T Cells--Size
Variation of Soluble Multimerization Reagent and Addition of
LCD8-Fab Addition for Stimulation
[0206] In this Example, the expansion of purified CD8+ T responder
cells stimulated in vitro with .alpha.CD3/.alpha.CD28 Fab fragments
that were reversibly immobilized soluble oligomeric streptavidin
muteins were examined In addition, the effect of adding
.alpha.CD8-Fab to the multimerization reagent for increasing the
specificity of the expansion for CD8+ T cells was examined
[0207] For this purpose, 300.000 purified CD8+ responder T cells
(Tresp) were separately stimulated with two different Streptactin
based multimerization reagents, namely either the small oligomeric
Streptactin multimerization reagent of Example 5 (1 mg/ml) or the
larger Streptactin oligomers described above (0.1 mg/ml). 3 .mu.l
of both different multimerization reagent (backbones) were either
loaded with a combination of the 0.5 .mu.g .alpha.CD3 and 0.5 .mu.g
.alpha.CD28 Fab fragments described above. In addition, 4.5 .mu.l
of the smaller Streptactin multimerization reagent (backbone) was
loaded with 0.5 .mu.g .alpha.CD3, 0.5 .mu.g .alpha.CD8 Fab and 0.5
.mu.g .alpha.CD28 Fab fragments described above. Furthermore 3
.mu.l of the "smaller" Streptactin multimerization reagent
(backbone) only functionalized with 0.5 .mu.g .alpha.CD3 Fab
fragment alone or 0.5 .mu.g .alpha.CD28 Fab fragment alone was
used. Unstimulated Tresp cells served as negative control and Tresp
stimulated with Dynabeads served as positive control. Tresp cells
were seeded in duplicates in 48-well plates in 1 ml cell culture
medium supplemented with 30 U/ml IL-2. Cells were incubated at
37.degree. C. with media exchange after 3 days and analyzed after 6
days. FIG. 20A depicts the degree of proliferation according to the
number of cells harvested at day 6 compared to the negative
controls and normalized to the positive control. FIG. 20A shows
that the expansion of the CD8+ T cells using the soluble
multimerization reagents of the invention result in higher yields
of the CD8+ T cells than expansion using dynabeads. The FACS
analysis of CD8 surface expression (FIG. 20B) and CD45RO surface
expression (FIG. 20C) after cell culture shows that the same
phenotype of CD8+ T cells were expanded by either the
multimerization reagents of the invention or Dynabeads (the various
stimulating conditions were compared using one-way ANOVA and no
significant difference (n.s.) was detected). The improved yield of
the CD8+ cells using the inventive expansion methods compared to
the Dynabeads might be due to the fact that the soluble
multimerization reagent can access their target receptors on the
cell surface better than the antibodies that are immobilized on the
Dynabeads. This improved yield might become very advantageous when
expanding rare population of cells from an initial sample.
[0208] In addition, comparing the yield of expansion achieved with
the multimerization agent on which both the 0.5 .mu.g .alpha.CD3
and 0.5 .mu.g .alpha.CD28 Fab fragments were jointly immobilized
(second column from the left in FIG. 20B) to the yield using two
multimerisation reagents which were functionalized only with the
.alpha.CD3 Fab fragment alone or the .alpha.CD28 Fab fragment alone
(third column from the left in FIG. 20B), it can be seen that both
experiments had the same expansion efficiency. Thus, these
experiments show that using one multimerization reagent on which
both the first agent and the second agent are jointly immobilized
is functionally equivalent to using for the expansion two separate
multimerization reagents which are loaded with only the first agent
and the second agent, respectively.
Example 14: Yield & Phenotype of Expanded CD8+ T
Cells--Titration of Separate Soluble Multimerization Reagents with
Different Ratios of LCD3- and .alpha.CD28 Fab Fragment Immobilized
Thereon
[0209] In this Example the yield and the phenotype of expanded CD8+
T responder cells (Tresp) that were stimulated in vitro with
.alpha.CD3/.alpha.CD28 Fab fragments that were reversibly
immobilized in different amounts on soluble oligomeric streptavidin
muteins were examined.
[0210] For this purpose 300.000 CD8+ responder T cells (Tresp) were
stimulated with varying amounts of a mixture of preparations of the
"small" oligomeric Streptactin multimerization reagent (1 mg/ml)
functionalized with .alpha.CD3 Fab alone and .alpha.CD28 Fab alone
("1.times." corresponds to 1.5 .mu.g Streptactin multimerization
reagent functionalized with 0.5 .mu.g .alpha.CD3 alone and 1.5
.mu.g multimerized Streptactin functionalized with 0.5 .mu.g
.alpha.CD28 Fab fragment alone), or 3 .mu.l of a preparation of the
Streptactin multimerization reagent loaded with 0.5 .mu.g
.alpha.CD3 and a0.5 .mu.g CD28 Fab, or 4.5 .mu.l of a preparation
of the Streptactin multimerization reagent loaded with 0.5 .mu.g
.alpha.CD3, 0.5 .mu.g strep-tagged .alpha.CD8 and 0.5 .mu.g
.alpha.CD28 Fab. Untreated Tresp cells served as negative control
and Tresp stimulated with Dynabeads as positive control. Tresp
cells were seeded in 48-well plates in 1 ml cell culture medium
supplemented with 30 U/ml IL-2. Cells were incubated at 37.degree.
C. without media exchange and analyzed after 5 days. FIG. 21A
depicts the degree of proliferation according to the number of
cells harvested at day 5 compared to the negative controls and
normalized to the positive control. FIG. 21A shows that the
expansion of the CD8+ T cells using the various soluble
multimerization reagents of the invention result in higher yields
of the CD8+ T cells than expansion using dynabeads (especially the
cumulative total reagent amount of the 5.times. condition resulted
in an optimal expansion of cells especially over time/increase in
total cells by beginning cell division). The FACS analysis of CD8
surface expression (FIG. 21B) and CD45RO surface expression (FIG.
21C) after cell culture shows that the same phenotype of CD8+ T
cells were expanded by either the various multimerization reagents
of the invention or by the commercially available Dynabeads.
Example 15: Activation of Intracellular Signaling Cascades After
Streptamer Multimers Stimulation of .alpha.CD19-CAR Transduced
Jurkat Cells
[0211] In this Example the activation of intracellular signaling
cascades of transduced Jurkat cells that have been modified to
express a tumor-specific chimeric antigen receptor (CAR), namely
here CD19 and that were stimulated using the oligomeric
Strep-tactin.RTM. of Example 5 as soluble multimerization reagent
was examined.
[0212] For this purpose, 300.000 Jurkat responder cells (Jresp)
were stimulated with (A) varying amounts of a mixture of
preparations of Streptactin multimerization reagent (1 mg/ml)
functionalized with .alpha.CD3 Fab and .alpha.CD28 Fab fragments
described here ("x1" corresponds to 3 .mu.g Streptactin
multerization reagent functionalized with 0.5 .mu.g .alpha.CD3- and
0.5 .mu.g .alpha.CD28 Fab--this provides a "polyclonal Streptactin
based multimerization reagent"), or (B) 3 .mu.l of a preparation of
Streptactin multimerization reagent functionalized with 0.5 .mu.g
(x1) or 1 .mu.g (x2) of the extracellular domain (ECD) of CD19 (the
natural ligand for the .alpha.CD19-CAR--this provides a
"CAR-specific Streptactin based multimerization reagent"), or 3
.mu.l of a preparation of Streptactin multimerization reagent
loaded with 0.5 .mu.g (x1) or 1 .mu.g (x2) .alpha.IgG recognizing
the IgG4 spacer within the .alpha.CD19-CAR--this also provides a
"CAR-specific Streptavidin mutein based multimerization reagent).
ECD of CD19 equipped with a hexahistidine tag was obtained from
Sino Biological/Life technologies (SEQ ID NO: 27) and was
functionalized for binding to the streptavidin based
multimerization reagent by mixing the ECD of CD19 with the adapter
molecule His-STREPPER (IBA GmbH, Germany, Order number 2-0920-005)
at a molecular ratio of 1:1 and incubating for 15 min at room
temperature. The His-STREPPER adapter molecule contains a chelating
portion that binds to the hexahistidine tag and a streptavidin
binding peptide, thereby temporarily providing the target molecule,
here the ECD of CD19 with a streptavidin binding peptide that can
reversibly bind to a streptavidin mutein based multimerization
reagent. Jresp stimulated with Dynabeads (beads having irreversibly
immobilized thereon .alpha.CD3- and .alpha.CD28- monoclonal
antibodies) or PMA and Ionomycin served as positive controls. Jresp
cells were seeded in 1.5 ml Eppendorf tubes in 200 .mu.lcell
culture medium supplemented with 30 U/ml IL-2. Cells were incubated
at 37.degree. C. and put on ice and lysed after 0 min to 20 min of
stimulation. Detection of phosphorylated ERK indicates active MAPK
signaling, staining of the housekeeper .beta.-Actin indicates
loading of equal amounts of total protein per condition and time
point. As can be seen from the comparison of FIG. 22A showing
activation of the Jurkat cells via the "polyclonal Streptactin
multimerization reagent" and FIG. 22B showing activation of the
Jurkat cells via the two "CAR-specific Streptactin based
multimerization reagents", the Jurkat cells can be
activated/expanded via the binding of the CD19 extracellular domain
to the CD19 specific chimeric antigen receptor. Since genetic
down-stream processing of T cells is almost exclusively performed
on pre-selected cell populations, a generic activation via
cross-linking of introduced CARs via the IgG4 spacer domain (this
is conserved within various CARs with different specificities)
broadens the applicability for reversible cell
stimulation/expansion in these in vitro cell-processing
situations.
[0213] Thus, this experiment shows that in principle any cell
population that is activated by binding of an agent (ligand) that
provides a primary activation signal to the cell population can be
expanded using a first agent reversibly immobilized on a
multimerization reagent as described here.
Example 16: Yield and Subset Composition of Expanded CD3+ T Cells
with Addition of LCD8-Fab for Stimulation
[0214] The experiment shows the expansion of purified CD3.sup.+ T
responder cells stimulated in vitro with .alpha.CD3/.alpha.CD28 Fab
fragments that were reversibly immobilized on the soluble
oligomeric Strep-tactin.RTM. of Example 5 that served a soluble
multimerization reagent. In one experiment, in addition to
.alpha.CD3/.alpha.CD28 Fab fragments, also a .alpha.CD8 Fab
fragment commercially available from IBA GmbH, Gottingen, Germany
(catalogue number 6-8000-203) was immobilized on the soluble
oligomer of the streptavidin mutein in order to test whether it is
possible to preferentially stimulate a specific T cell
subpopulation in vitro with the reversible .alpha.CD3/.alpha.CD28
Fab-Streptamer multimers. In more detail, 500.000 purified
CD3.sup.+ responder T cells (Tresp) were stimulated with 3 .mu.l of
a preparation of oligomeric Streptavidin (1 mg/ml) loaded with a
combination of 0.5 .mu.g of the .alpha.CD3 and 0.5 .mu.g of the
.alpha.CD28 Fab. As an alternative approach, 4.5 .mu.l of the
Streptactin oligomer were loaded with 0.5 .mu.g .alpha.CD3, 0.5
.mu.g strep-tagged .alpha.CD8 Fab and 0.5 .mu.g strep-tagged
.alpha.CD28 Fab. Unstimulated Tresp cells served as negative
control and Tresp stimulated with Dynabeads (beads on which
.alpha.CD3 and .alpha.CD28 monoclonal antibodies are irreversible
immobilized) served as positive control. As can be seen from FIG.
23A, the multimerization reagent that is reversibly loaded with the
.alpha.CD3 Fab fragment, the .alpha.CD28 Fab fragment and also the
.alpha.CD8 Fab fragment provided the highest number of expanded
CD3+ T cells. With 1.times.1.times.10.sup.6 the number of expanded
cells the yield was about 30% higher than for expansion of these T
cells using commercially available Dynabeads. In addition and more
important, as shown in FIG. 23B with this multimerization reagent
that caries the .alpha.CD3 Fab fragment, the .alpha.CD28 Fab
fragment and the .alpha.CD8 Fab fragment, the amount of CD8+ T
cells were the highest, compared to both the expansion with
Dynabeads or a soluble multimerization reagent of the invention
that caries only the .alpha.CD3 Fab fragment and the .alpha.CD28
Fab fragment as first and second agent as described herein. Thus,
also this experiment shows the advantage of the present invention
that in addition to a first agent that provides a primary
activation signal to the desired cell population and optionally a
second agent that provide a co-stimulatory signal, a further agent
that is specific for the activation of the desired cell population
can be immobilized on the multimerization reagent. Thus, by so
doing, the present invention provides for the possibility to
preferentially expand or selectively enrich any desired cell
(sub)population from an sample that, for example, comprises a
variety of different subpopulations.
Example 17: Parallel Antigen-Specific Expansion of Tcm Responder
Cells Out of a Single Pool
[0215] In this Example, the kinetics of parallel Antigen specific
(Ag-specific) expansion out of a single pool of T responder cells
stimulated in vitro with multiple reversible
peptide:MHC/.alpha.CH28 Fab-Streptamer multimers is examined
500.000 CD3+CD62L+CD45RA- responder Tcm cells (Tresp) are
simultaneously stimulated for multiple Ag-specificities using for
each specificity, 3 .mu.l of Streptactin multimers functionalized
with 0.5 .mu.g of the respective peptide:MHC class I complexes that
carries a streptavidin binding peptide and 0.5 .mu.g .alpha.CD28
Fab that also carries a streptavidin binding peptide. As an
alternative approach, 4.5 .mu.l of Streptactin based
multimerization reagent functionalized with 0.5 .mu.g peptide:MHC
class I complexes carrying a streptavidin binding peptide, 0.5
.mu.g .alpha.CD8 Fab and 0.5 .mu.g .alpha.CD28 Fab as described
here are used for each specificity. For comparison, polyclonal
stimulation is performed, using 3 .mu.l of a preparation of
Streptactin based multimerization reagent (1 mg/ml) either
reversibly loaded with a combination of 0.5 .mu.g .alpha.CD3 Fab
and 0.5 .mu.g .alpha.CD28 Fab. Again as the alternative stimulation
condition described above, 4.5 .mu.l of a preparation of the
Streptactin based multimerization reagent reversibly loaded with
0.5 .mu.g .alpha.CD3 Fab, 0.5 .mu.g .alpha.CD8 Fab and 0.5 .mu.g
.alpha.CD28 Fab (each of them carrying a streptavidin binding
peptide can be used. Untreated (unstimulated) Tresp cells serve as
negative control and Tresp cells stimulated polyclonal with
Dynabeads (.alpha.CD3- and .alpha.CD28- mAb coated beads) as
positive control. Tresp cells are seeded in 48-well plates in lml
cell culture medium supplemented with 30 U/ml IL-2 and 5ng/ml
IL-15. Cells are incubated at 37.degree. C. with media exchange
every 3 days and cell count are analyzed after 7 and 14 days.
Example 18: Preferential Proliferation of CD8+ T Cells Among CD3+ T
Responder Cells Stimulated In Vitro with Streptavidin Based
Multimerization Reagents Reversibly Functionalized with
.alpha.CD3/.alpha.CD8/.alpha.CD28 Fab Fragments
[0216] 300.000 CD3+ responder T cells (Tresp) are stimulated with 3
.mu.l of a preparation of Streptactin multimerization (1 mg/ml) or
a preparation of a multimerization reagent using the large
Streptactin backbone (0.1 mg/ml) either loaded with a combination
of 0.5 .mu.g .alpha.CD3 and 0.5 .mu.g .alpha.CD28 Fab, or 4.5 .mu.l
of a preparation of Streptactin based multimerization reagent
loaded with 0.5 .mu.g .alpha.CD3, 0.5 .mu.g .alpha.CD8 Fab and 0.5
.mu.g aCD28 Fab, or 3 .mu.l of a mixture of preparations of
Streptactin based multimerization reagent with 0.5 .mu.g aCD3 Fab
alone and 0.5 .mu.g aCD28 Fab alone (each Fab fragment again
carries a streptavidin binding peptide). Untreated Tresp cells
serve as negative control and Tresp stimulated with Dynabeads
(aCD3- and aCD28- mAb coated beads) as positive control. Tresp
cells are seeded in duplicates in 48-well plates in 1 ml cell
culture medium supplemented with 30 U/ml IL-2. Cells are incubated
at 37.degree. C. with media exchange after 3 days and analyzed
after 6 days.
Example 19: Preferential Proliferation of CD8+ T Cells Among CD3+ T
Responder Cells Stimulated In VitrowWith Streptavidin Based
Multimerization Reagents Reversibly Functionalized with .alpha.CD3
and .alpha.CD28 Fab Fragments
[0217] 300.000 CD3+ responder T cells (Tresp) are stimulated with
varying amounts of a mixture of preparations of Streptactin based
multimerization reagent (1 mg/ml) functionalized with .alpha.CD3
Fab fragment alone and .alpha.CD28 Fab fragment alone (1.5 .mu.g
Streptactin based multimerization reagent functionalized with 0.5
.mu.g .alpha.CD3 Fab fragment alone and 1.5 .mu.g Streptactin based
multimerization reagent functionalized with 0.5 .mu.g .alpha.CD28
Fab fragment alone), or varying amounts of a mixture of
preparations of Streptactin based multimerization reagent
functionalized with .alpha.CD3 Fab fragment and .alpha.CD28 Fab
fragment with or without .alpha.CD8 Fab fragment (each Fab fragment
again carries a streptavidin binding peptide) (3 .mu.g Streptactin
based multimerization reagent functionalized with 0.5 .mu.g
.alpha.CD3- and 0.5 .mu.g .alpha.CD28 Fab fragment--without
.alpha.CD8 Fab fragment, or 4.5 .mu.l of a preparation of
Streptactin multimerization reagent loaded with 0.5 .mu.g
.alpha.CD3 Fab fragment, 0.5 .mu.g .alpha.CD8 Fab fragment and 0.5
.mu.g .alpha.CD28 Fab fragment, wherein Fab fragment again carries
a streptavidin binding peptide). Untreated Tresp cells serve as
negative control and Tresp stimulated with Dynabeads (.alpha.CD3-
and .alpha.CD28- mAb coated beads) as positive control. Tresp cells
are seeded in 48-well plates in 1 ml cell culture medium
supplemented with 30 U/ml IL-2. Cells are incubated at 37.degree.
C. with media exchange after 3 days and analyzed after 6 days.
[0218] The listing or discussion of a previously published document
in this specification should not necessarily be taken as an
acknowledgement that the document is part of the state of the art
or is common general knowledge.
[0219] The invention illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including," containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
exemplary embodiments and optional features, modification and
variation of the inventions embodied therein herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention.
[0220] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0221] Other embodiments are within the following claims. In
addition, where features or aspects of the invention are described
in terms of Markush groups, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
Sequence CWU 1
1
2718PRTartificialStrep-tag 1Trp Ser His Pro Gln Phe Glu Lys1
524PRTartificialstreptavidin mutein (analog) 1 2Val Thr Ala
Arg134PRTartificialstreptavidin mutein (analog) 2 3Ile Gly Ala
Arg1428PRTartificialdi-tag3 4Trp Ser His Pro Gln Phe Glu Lys Gly
Gly Gly Ser Gly Gly Gly Ser1 5 10 15Gly Gly Gly Ser Trp Ser His Pro
Gln Phe Glu Lys 20 25524PRTartificialdi-tag2 5Trp Ser His Pro Gln
Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser1 5 10 15Trp Ser His Pro
Gln Phe Glu Lys 2069PRTartificialimmediate-early 1 protein (amino
acids 309-317) 6Cys Arg Val Leu Cys Cys Tyr Val Leu1
5730PRTartificialTwin-Strep-tag 7Ser Ala Trp Ser His Pro Gln Phe
Glu Lys Gly Gly Gly Ser Gly Gly1 5 10 15Gly Ser Gly Gly Ser Ala Trp
Ser His Pro Gln Phe Glu Lys 20 25 3089PRTartificialpp65 epitope of
CMV (amino acids 341-350) 8Gln Tyr Asp Pro Val Ala Ala Leu Phe1
5911PRTartificialpp65 epitope of CMV (amino acids 265-274) 9Arg Pro
His Glu Arg Asn Gly Phe Thr Val Leu1 5 101011PRTartificialhexon 5
epitope of adenovirus (amino acids 114-124) 10Lys Pro Tyr Ser Gly
Thr Ala Tyr Asn Ala Leu1 5 10119PRTartificialHA-tag 11Tyr Pro Tyr
Asp Val Pro Asp Tyr Ala1 51211PRTartificialVSV-G-tag 12Tyr Thr Asp
Ile Glu Met Asn Arg Leu Gly Lys1 5 101311PRTartificialHSV-tag 13Gln
Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp1 5 101410PRTartificialT7
epitope 14Ala Ser Met Thr Gly Gly Gln Gln Met Gly1 5
101510PRTartificialmyc epitope 15Glu Gln Lys Leu Ile Ser Glu Glu
Asp Leu1 5 101614PRTartificialV5-tag 16Gly Lys Pro Ile Pro Asn Pro
Leu Leu Gly Leu Asp Ser Thr1 5 1017119PRTmus musculus 17Gln Val Gln
Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30Thr
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Thr Leu Thr Val Ser Ser
11518106PRTmus musculus 18Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala
Ser Ser Ser Val Ser Tyr Met 20 25 30Asn Trp Tyr Gln Gln Lys Ser Gly
Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser
Gly Val Pro Ala His Phe Arg Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr
Ser Leu Thr Ile Ser Gly Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr
Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95Phe Gly Ser
Gly Thr Lys Leu Glu Ile Asn 100 10519116PRTmus musculus 19Leu Gln
Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Arg1 5 10 15Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Ile Ile His 20 25
30Trp Ile Lys Leu Arg Ser Gly Gln Gly Leu Glu Trp Ile Gly Trp Phe
35 40 45Tyr Pro Gly Ser Asn Asp Ile Gln Tyr Asn Ala Lys Phe Lys Gly
Lys 50 55 60Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr Met
Glu Leu65 70 75 80Thr Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
Cys Ala Arg Arg 85 90 95Asp Asp Phe Ser Gly Tyr Asp Ala Leu Pro Tyr
Trp Gly Gln Gly Thr 100 105 110Met Val Thr Val 11520108PRTmus
musculus 20Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser
Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Thr Asn Glu Asn Ile
Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
Gln Leu Leu Ile 35 40 45Tyr Ala Ala Thr His Leu Val Glu Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys
Ile Thr Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly Asn Tyr Tyr Cys
Gln His Phe Trp Gly Thr Pro Cys 85 90 95Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg 100 10521314PRTartificialHLA-A*2402 21Met Gly
Ser His Ser Met Arg Tyr Phe Ser Thr Ser Val Ser Arg Pro1 5 10 15Gly
Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr 20 25
30Gln Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro
35 40 45Arg Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Glu
Glu 50 55 60Thr Gly Lys Val Lys Ala His Ser Gln Thr Asp Arg Glu Asn
Leu Arg65 70 75 80Ile Ala Leu Arg Tyr Tyr Asn Gln Ser Glu Ala Gly
Ser His Thr Leu 85 90 95Gln Met Met Phe Gly Cys Asp Val Gly Ser Asp
Gly Arg Phe Leu Arg 100 105 110Gly Tyr His Gln Tyr Ala Tyr Asp Gly
Lys Asp Tyr Ile Ala Leu Lys 115 120 125Glu Asp Leu Arg Ser Trp Thr
Ala Ala Asp Met Ala Ala Gln Ile Thr 130 135 140Lys Arg Lys Trp Glu
Ala Ala His Val Ala Glu Gln Gln Arg Ala Tyr145 150 155 160Leu Glu
Gly Thr Cys Val Asp Gly Leu Arg Arg Tyr Leu Glu Asn Gly 165 170
175Lys Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His
180 185 190His Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala
Leu Gly 195 200 205Phe Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg
Asp Gly Glu Asp 210 215 220Gln Thr Gln Asp Thr Glu Leu Val Glu Thr
Arg Pro Ala Gly Asp Gly225 230 235 240Thr Phe Gln Lys Trp Ala Ala
Val Val Val Pro Ser Gly Glu Glu Gln 245 250 255Arg Tyr Thr Cys His
Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr 260 265 270Leu Arg Trp
Glu Pro Pro Pro Ser Gly Ser Ser Ala Trp Ser His Pro 275 280 285Gln
Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 290 295
300Ser Ala Trp Ser His Pro Gln Phe Glu Lys305
31022314PRTartificialHLA-B*0702 22Met Gly Ser His Ser Met Arg Tyr
Phe Tyr Thr Ser Val Ser Arg Pro1 5 10 15Gly Arg Gly Glu Pro Arg Phe
Ile Ser Val Gly Tyr Val Asp Asp Thr 20 25 30Gln Phe Val Arg Phe Asp
Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro 35 40 45Arg Ala Pro Trp Ile
Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn 50 55 60Thr Gln Ile Tyr
Lys Ala Gln Ala Gln Thr Asp Arg Glu Ser Leu Arg65 70 75 80Asn Leu
Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu 85 90 95Gln
Ser Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg 100 105
110Gly His Asp Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn
115 120 125Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln
Ile Thr 130 135 140Gln Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln
Arg Arg Ala Tyr145 150 155 160Leu Glu Gly Glu Cys Val Glu Trp Leu
Arg Arg Tyr Leu Glu Asn Gly 165 170 175Lys Asp Lys Leu Glu Arg Ala
Asp Pro Pro Lys Thr His Val Thr His 180 185 190His Pro Ile Ser Asp
His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly 195 200 205Phe Tyr Pro
Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp 210 215 220Gln
Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg225 230
235 240Thr Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu
Gln 245 250 255Arg Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys
Pro Leu Thr 260 265 270Leu Arg Trp Glu Pro Pro Pro Ser Gly Ser Ser
Ala Trp Ser His Pro 275 280 285Gln Phe Glu Lys Gly Gly Gly Ser Gly
Gly Gly Ser Gly Gly Gly Ser 290 295 300Ser Ala Trp Ser His Pro Gln
Phe Glu Lys305 31023321PRTartificialHLA-C*0702 23Met Ala Leu Thr
Glu Thr Trp Ala Cys Ser His Ser Met Arg Tyr Phe1 5 10 15Asp Thr Ala
Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ser 20 25 30Val Gly
Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala 35 40 45Ala
Ser Pro Arg Gly Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly 50 55
60Pro Glu Tyr Trp Asp Arg Glu Thr Gln Lys Tyr Lys Arg Gln Ala Gln65
70 75 80Ala Asp Arg Val Ser Leu Arg Asn Leu Arg Gly Tyr Tyr Asn Gln
Ser 85 90 95Glu Asp Gly Ser His Thr Leu Gln Arg Met Ser Gly Cys Asp
Leu Gly 100 105 110Pro Asp Gly Arg Leu Leu Arg Gly Tyr Asp Gln Ser
Ala Tyr Asp Gly 115 120 125Lys Asp Tyr Ile Ala Leu Asn Glu Asp Leu
Arg Ser Trp Thr Ala Ala 130 135 140Asp Thr Ala Ala Gln Ile Thr Gln
Arg Lys Leu Glu Ala Ala Arg Ala145 150 155 160Ala Glu Gln Leu Arg
Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu 165 170 175Arg Arg Tyr
Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Ala Glu Pro 180 185 190Pro
Lys Thr His Val Thr His His Pro Leu Ser Asp His Glu Ala Thr 195 200
205Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Thr Leu Thr
210 215 220Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu Leu
Val Glu225 230 235 240Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys
Trp Ala Ala Val Val 245 250 255Val Pro Ser Gly Gln Glu Gln Arg Tyr
Thr Cys His Met Gln His Glu 260 265 270Gly Leu Gln Glu Pro Leu Thr
Leu Ser Trp Glu Pro Ser Ser Gln Pro 275 280 285Thr Ile Gly Ser Ala
Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly 290 295 300Ser Gly Gly
Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu305 310 315
320Lys24100PRThomo sapiens 24Met Ile Gln Arg Thr Pro Lys Ile Gln
Val Tyr Ser Arg His Pro Ala1 5 10 15Glu Asn Gly Lys Ser Asn Phe Leu
Asn Cys Tyr Val Ser Gly Phe His 20 25 30Pro Ser Asp Ile Glu Val Asp
Leu Leu Lys Asn Gly Glu Arg Ile Glu 35 40 45Lys Val Glu His Ser Asp
Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr 50 55 60Leu Leu Tyr Tyr Thr
Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala65 70 75 80Cys Arg Val
Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp 85 90 95Asp Arg
Asp Met 10025118PRTmus musculus 25Gln Val Thr Leu Lys Glu Ser Gly
Pro Gly Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser
Phe Ser Gly Phe Ser Leu Arg Thr Ser 20 25 30Gly Met Gly Val Gly Trp
Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val65 70 75 80Phe Leu
Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys
Ala Gln Ile Asn Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 11526111PRTmus musculus 26Asp Ile Val
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg
Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Phe Asp 20 25 30Gly
Asp Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45Lys Leu Leu Ile Tyr Thr Thr Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60Arg Phe Ser Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
His65 70 75 80Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln
Gln Ser Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 11027283PRTartificialhuman CD19 extracellular
domain with His-Tag 27Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu
Gly Asp Asn Ala Val1 5 10 15Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly
Pro Thr Gln Gln Leu Thr 20 25 30Trp Ser Arg Glu Ser Pro Leu Lys Pro
Phe Leu Lys Leu Ser Leu Gly 35 40 45Leu Pro Gly Leu Gly Ile His Met
Arg Pro Leu Ala Ile Trp Leu Phe 50 55 60Ile Phe Asn Val Ser Gln Gln
Met Gly Gly Phe Tyr Leu Cys Gln Pro65 70 75 80Gly Pro Pro Ser Glu
Lys Ala Trp Gln Pro Gly Trp Thr Val Asn Val 85 90 95Glu Gly Ser Gly
Glu Leu Phe Arg Trp Asn Val Ser Asp Leu Gly Gly 100 105 110Leu Gly
Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser Pro 115 120
125Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp Arg
130 135 140Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro Arg
Asp Ser145 150 155 160Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met
Ala Pro Gly Ser Thr 165 170 175Leu Trp Leu Ser Cys Gly Val Pro Pro
Asp Ser Val Ser Arg Gly Pro 180 185 190Leu Ser Trp Thr His Val His
Pro Lys Gly Pro Lys Ser Leu Leu Ser 195 200 205Leu Glu Leu Lys Asp
Asp Arg Pro Ala Arg Asp Met Trp Val Met Glu 210 215 220Thr Gly Leu
Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala Gly Lys Tyr225 230 235
240Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu Glu Ile Thr
245 250 255Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly Gly
Trp Lys 260 265 270Ala His His His His His His His His His His 275
280
* * * * *