U.S. patent application number 17/062331 was filed with the patent office on 2021-02-04 for methods for manufacturing t cells.
The applicant listed for this patent is Immatics US, Inc.. Invention is credited to Amir Alpert, Agathe Bourgogne, Zoe Coughlin, Mamta KALRA, Ali Mohamed, Steffen Walter.
Application Number | 20210030803 17/062331 |
Document ID | / |
Family ID | 1000005153978 |
Filed Date | 2021-02-04 |
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United States Patent
Application |
20210030803 |
Kind Code |
A1 |
KALRA; Mamta ; et
al. |
February 4, 2021 |
METHODS FOR MANUFACTURING T CELLS
Abstract
The disclosure relates to methods of manufacturing T cells for
adoptive immunotherapy. The disclosure further provides for methods
of genetically transducing T cells, methods of using T cells, and T
cell populations thereof. In an aspect, the disclosure provides for
methods of thawing frozen peripheral blood mononuclear cells
(PBMC), resting the thawed PBMC, activating the T cell in the
cultured PBMC with an anti-CD3 antibody and an anti-CD28 antibody
immobilized on a solid phase, transducing the activated T cell with
a viral vector, expanding the transduced T cell, and obtaining
expanded T cells.
Inventors: |
KALRA; Mamta; (Sugar Land,
TX) ; Coughlin; Zoe; (Richmond, TX) ; Alpert;
Amir; (Houston, TX) ; Walter; Steffen;
(Houston, TX) ; Mohamed; Ali; (Sugar Land, TX)
; Bourgogne; Agathe; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immatics US, Inc. |
Houston |
TX |
US |
|
|
Family ID: |
1000005153978 |
Appl. No.: |
17/062331 |
Filed: |
October 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16271393 |
Feb 8, 2019 |
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17062331 |
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62628521 |
Feb 9, 2018 |
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62647571 |
Mar 23, 2018 |
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62633113 |
Feb 21, 2018 |
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62726350 |
Sep 3, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/86 20130101;
A61K 35/17 20130101; C12N 2501/2321 20130101; C12N 2501/515
20130101; C12N 2501/51 20130101; C12N 5/0637 20130101; C12N
2501/2312 20130101; A61P 35/00 20180101; C12N 2510/00 20130101;
C12N 2501/2307 20130101; C12N 5/0638 20130101; C12N 2501/2315
20130101; C07K 16/2818 20130101; C07K 16/2809 20130101; A61P 31/00
20180101; C12N 5/0636 20130101; C12N 2501/2302 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C12N 5/0783 20060101 C12N005/0783; C07K 16/28 20060101
C07K016/28; C12N 15/86 20060101 C12N015/86; A61P 31/00 20060101
A61P031/00; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 2018 |
DE |
10 2018 102 971.3 |
Feb 28, 2018 |
DE |
10 2018 104 628.6 |
Apr 16, 2018 |
DE |
10 2018 108 996.1 |
Claims
1. A method of transducing a T cell population comprising thawing
frozen peripheral blood mononuclear cells (PBMC), resting the
thawed PBMC, activating the T cells in the rested PBMC with an
anti-CD3 antibody and anti-CD28 antibody, transducing the activated
T cells with a viral vector, expanding the transduced T cells, and
obtaining the expanded T cells, wherein the expanded T cells are
capable of specifically binding a peptide consisting of the amino
acid sequence of SLLMWITQC (SEQ ID NO: 131) or GVYDGREHTV (SEQ ID
NO: 89).
2. The method of claim 1, wherein the activation further comprises
incubation with IL-2.
3. The method of claim 1, wherein the IL-2 concentration is between
about 50 IU/mL and 150 IU/mL.
4. The method of claim 1, wherein the viral vector is a lentivirus
vector.
5. The method of claim 1, wherein the T cells are expanded for 1-15
days.
6. The method of claim 1, wherein the T cells are expanded in the
presence of IL-2, IL-7, IL-12, IL-15, or a combination thereof.
7. The method of claim 1, wherein the T cells are CD4+.
8. The method of claim 1, wherein the T cells are CD8+.
9. A method of treating a patient having a cancer comprising
administering a composition comprising the T cell of claim 1.
10. The method of claim 9, wherein the cancer is melanoma, ovarian
cancer, esophageal cancer, non-small cell lung cancer (NSCLC), or a
combination thereof.
11. The method of claim 9, the T cells are autologous.
12. The method of claim 9, wherein the patient is HLA-A*02.
13. The method of claim 9, wherein the dosage of the T-cells is
about 1.times.10.sup.6 to about 1.times.10.sup.9 transduced T
cells/m.sup.2 (or kg) of the patient.
14. The method of claim 9, wherein the T-cells are administered via
continuous infusion.
15. The method of claim 1, wherein the expanded T cells are capable
of specifically binding a peptide consisting of the amino acid
sequence of SLLMWITQC (SEQ ID NO: 131).
16. The method of claim 15, wherein the viral vector comprises a
nucleic acid encoding a T cell receptor (TCR) that binds a peptide
consisting of the amino acid sequence of SLLMWITQC (SEQ ID NO:
131).
17. The method of claim 1, wherein the expanded T cells are capable
of specifically binding a peptide consisting of the amino acid
sequence of GVYDGREHTV (SEQ ID NO: 89).
18. The method of claim 17, wherein the viral vector comprises a
nucleic acid encoding a TCR that binds a peptide consisting of the
amino acid sequence of GVYDGREHTV (SEQ ID NO: 89).
19. The method of claim 6, wherein the T cells are expanded in the
presence of IL-7.
20. The method of claim 6, wherein the T cells are expanded in the
presence of IL-15.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/271,393, filed 8 Feb. 2019, which claims
priority to U.S. Provisional application No. 62/726,350, filed on
Sep. 3, 2018, U.S. provisional application No. 62/647,571, filed on
Mar. 23, 2018, U.S. provisional application No. 62/633,113, filed
on Feb. 21, 2018, U.S. provisional application No. 62/628,521,
filed on Feb. 9, 2018, German Patent Application number 10 2018 108
996.1, filed Apr. 16, 2018; German Patent Application number 10
2018 104 628.6, filed Feb. 28, 2018; and German Patent Application
number 10 2018 102 971.3, filed Feb. 9, 2018, the contents of each
are hereby incorporated by reference in their entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT
FILE (.TXT)
[0002] Pursuant to the EFS-Web legal framework and 37 CFR
.sctn..sctn. 1.821-825 (see MPEP .sctn. 2442.03(a)), a Sequence
Listing in the form of an ASCII-compliant text file (entitled
"Sequence_Listing_3000011-006005_ST25.txt" created on 1 Oct. 2020,
and 24,767 bytes in size) is submitted concurrently with the
instant application, and the entire contents of the Sequence
Listing are incorporated herein by reference.
FIELD
[0003] The present disclosure generally relates to methods of
manufacturing T cells for adoptive immunotherapy. The disclosure
further provides for methods of genetically transducing T cells,
methods of using T cells, and T cell populations thereof.
BACKGROUND
[0004] Redirecting the specificity of T cells against
tumor-associated antigens by genetically enforced expression of T
cell receptors (TCRs) or chimeric antigen receptor (CARs) has
recently boosted the field of adoptive T cell transfer. The use of
second- and third-generation CARs has helped to resolve the
long-standing problem of insufficient in vivo T cell persistence
after transfer that was severely hampering its efficacy.
Nevertheless, important obstacles for a wider application remain,
such as the necessity to produce T cell products on an
individualized basis, making this promising treatment approach
hardly economically feasible. Although the use of T cells, for
example autologous T cells, has shown promise, it can be difficult
to obtain a suitable numbers of autologous cells in heavily
pretreated patients.
[0005] U.S. 2003/0170238 and U.S. 2003/0175272 describe methods for
adoptive immunotherapy, in which T cells are allowed to rest by
removing them from activation stimuli for at least 48-72 hours,
typically at least about 72-120 hours, and then reactivating them
prior to infusion by labeling cells, for example, with mitogenic
monoclonal antibodies (mAbs), such as soluble anti-CD3 and
anti-CD28 mAbs, and then mixing the labeled cells with autologous
mononuclear cells that are optionally enhanced in monocytes and
granulocytes.
[0006] U.S. 2017/0051252 describes methods for manufacturing T cell
therapeutics including the steps of obtaining a population of cells
containing T cells and antigen presenting cells (APCs); culturing
the population of cells in a cell culture medium comprising (i) one
or more cytokines, (ii) an anti-CD3 antibody or CD3-binding
fragment thereof, and (iii) an anti-CD28 antibody or a CD28-binding
fragment thereof, B7-1 or a CD28-binding fragment thereof, or B7-2
or a CD28-binding fragment thereof, in which the culture activates
and stimulates the T cells; transducing the population of activated
cells with a viral vector; and culturing the population of cells in
a cell growth medium to expand the transduced T cells; thereby
manufacturing T cell therapeutics.
[0007] Improved strategies are needed for transducing cell
populations in vitro that could generate enough T cells for
research, diagnostic, and therapeutic purposes. A solution to this
technical problem is provided herein.
BRIEF SUMMARY
[0008] In an aspect, the present disclosure relates to a method of
transducing a T cell including thawing frozen peripheral blood
mononuclear cells (PBMC), resting the thawed PBMC, activating the T
cell in the cultured PBMC with an anti-CD3 antibody and an
anti-CD28 antibody, transducing the activated T cell with a viral
vector, expanding the transduced T cell, and obtaining the expanded
T cells.
[0009] In an aspect, the T cell is activated in cultured PBMC with
an anti-CD3 antibody and an anti-CD28 antibody immobilized on a
solid phase support.
[0010] In another aspect, the resting step may be carried out
within a period of no more than about 1 hour, no more than about 2
hours, no more than about 3 hours, no more than about 4 hours, no
more than about 5 hours, no more than about 6 hours, no more than
about 7 hours, no more than about 8 hours, no more than about 9
hours, no more than about 10 hours, no more than about 11 hours, no
more than about 12 hours, no more than about 18 hours, no more than
about 24 hours, no more than about 48 hours, no more than about 36
hours, no more than about 48 hours, no more than about 60 hours, no
more than about 72 hours, no more than about 84 hours, no more than
about 96 hours, no more than about 108 hours, or no more than about
120 hours.
[0011] In another aspect, resting may be carried out within a
period of from about 0.5 hour to about 48 hours, about 0.5 hour to
about 36 hours, about 0.5 hour to about 24 hours, about 0.5 hour to
about 18 hours, about 0.5 hour to about 12 hours, about 0.5 hour to
about 6 hours, about 1 hour to about 6 hours, about 2 hours to
about 5 hours, about 3 hours to about 5 hours, about 3 hours to
about 4 hours, about 4 to about 5 hours, or about 1 hours to about
24 hours, about 2 to about 24 hours, about 12 to about 48 hours,
about 0.5 hour to about 120 hours, about 0.5 hour to about 108
hours, about 0.5 hour to about 96 hours, about 0.5 hour to about 84
hours, about 0.5 hour to about 72 hours, or about 0.5 hour to about
60 hours.
[0012] In another aspect, the resting step may be carried out
within a period of about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, or about 10 hours.
[0013] In an aspect, the fold expansion of T cells produced with a
resting step of about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 10 hours, about 2 hours to about 5 hours,
about 3 hours to about 5 hours, about 3 hours to about 4 hours, or
about 4 to about 5 hours is about equal to (about 1:1); about at
least 1.1 times, about at least 1.2 times, about at least 1.3
times, about at least 1.5 times, about at least 1.7 times, or about
at least 2.0 times greater than the fold expansion of T cells
produced with a resting step of about 16 hours, about 17 hours,
about 18 hours, about 19 hours, about 20 hours, about 24 hours, or
about 16 to about 20 hours. In a preferred aspect, the fold
expansion of T cells produced with a resting step of about 4 hours
is about at least 1.5 times greater than the fold expansion of T
cells produced with a resting step of about 16 hours (for example,
overnight). In an aspect, the only difference between the
production of the T cells is the reduced resting time.
[0014] In an aspect, the number of T cells produced with a resting
step of about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9
hours, about 10 hours, about 2 hours to about 5 hours, about 3
hours to about 5 hours, about 3 hours to about 4 hours, or about 4
to about 5 hours is about equal to (about 1:1); about at least 1.1
times, about at least 1.2 times, about at least 1.3 times, about at
least 1.5 times, about at least 1.7 times, or about at least 2.0
times greater than the number of T cells produced with a resting
step of about 16 hours, about 17 hours, about 18 hours, about 19
hours, about 20 hours, about 24 hours, or about 16 to about 20
hours. In a preferred aspect, the number of T cells produced with a
resting step of about 4 hours is about at least 1.5 times or about
1.3 times to about 2.0 times greater than the fold expansion of T
cells produced with a resting step of about 16 hours (for example,
overnight). In an aspect, the only difference between the
production of the T cells is the reduced resting time.
[0015] In yet another aspect, anti-CD3 antibody and the anti-CD28
antibody each may have a concentration of no more than about 0.1
.mu.g/ml, no more than about 0.2 .mu.g/ml, no more than about 0.3
.mu.g/ml, no more than about 0.4 .mu.g/ml, no more than about 0.5
.mu.g/ml, no more than about 0.6 .mu.g/ml, no more than about 0.7
.mu.g/ml, no more than about 0.8 .mu.g/ml, no more than about 0.9
.mu.g/ml, no more than about 1.0 .mu.g/ml, no more than about 2.0
.mu.g/ml, no more than about 4.0 .mu.g/ml, no more than about 6.0
.mu.g/ml, no more than about 8.0 .mu.g/ml, or no more than about
10.0 .mu.g/ml.
[0016] In yet another aspect, anti-CD3 antibody and the anti-CD28
antibody each may have a concentration of from about 0.1 .mu.g/ml
to about 1.0 .mu.g/ml, about 0.1 .mu.g/ml to about 0.8 .mu.g/ml,
about 0.1 .mu.g/ml to about 0.6 .mu.g/ml, about 0.1 .mu.g/ml to
about 0.5 .mu.g/ml, about 0.1 .mu.g/ml to about 0.25 .mu.g/ml,
about 0.2 .mu.g/ml to about 0.5 .mu.g/ml, about 0.2 .mu.g/ml to
about 0.3 .mu.g/ml, about 0.3 .mu.g/ml to about 0.5 .mu.g/ml, about
0.3 .mu.g/ml to about 0.4 .mu.g/ml, about 0.2 .mu.g/ml to about 0.5
.mu.g/ml, about 0.1 .mu.g/ml to about 10.0 .mu.g/ml, about 0.1
.mu.g/ml to about 8.0 .mu.g/ml, about 0.1 .mu.g/ml to about 6.0
.mu.g/ml, about 0.1 .mu.g/ml to about 4.0 .mu.g/ml, or about 0.1
.mu.g/ml to about 2.0 .mu.g/ml.
[0017] In an aspect, activation described herein may be carried out
within a period of no more than about 1 hour, no more than about 2,
hours, no more than about 3 hours, no more than about 4 hours, no
more than about 5 hours, no more than about 6 hours, no more than
about 7 hours, no more than about 8 hours, no more than about 9
hours, no more than about 10 hours, no more than about 11 hours, no
more than about 12 hours, no more than about 14 hours, no more than
about 16 hours, no more than about 18 hours, no more than about 20
hours, no more than about 22 hours, no more than about 24 hours, no
more than about 26 hours, no more than about 28 hours, no more than
about 30 hours, no more than about 36 hours, no more than about 48
hours, no more than about 60 hours, no more than about 72 hours, no
more than about 84 hours, no more than about 96 hours, no more than
about 108 hours, or no more than about 120 hours.
[0018] In another aspect, activation described herein may be
carried out within a period of from about 1 hour to about 120
hours, about 1 hour to about 108 hours, about 1 hour to about 96
hours, about 1 hour to about 84 hours, about 1 hour to about 72
hours, about 1 hour to about 60 hours, about 1 hour to about 48
hours, about 1 hour to about 36 hours, about 1 hour to about 24
hours, about 2 hours to about 24 hours, about 4 hours to about 24
hours, about 6 hours to about 24 hours, about 8 hours to about 24
hours, about 10 hours to about 24 hours, about 12 hours to about 24
hours, about 12 hours to about 72 hours, about 24 hours to about 72
hours, about 6 hours to about 48 hours, about 24 hours to about 48
hours, about 6 hours to about 72 hours, or about 1 hours to about
12 hours.
[0019] In an aspect, T cells described herein are autologous to the
patient or individual. In another aspect, T cells described herein
are allogenic to the patient or individual.
[0020] In another aspect, a solid phase described herein may be a
surface of a bead, a plate, a flask, or a bag.
[0021] In yet another aspect, a plate described herein may be a
6-well, 12-well, or 24-well plate.
[0022] In an aspect, a flask described herein may have a seeding
surface area of at least about 25 cm.sup.2, about 75 cm.sup.2,
about 92.6 cm.sup.2, about 100 cm.sup.2, about 150 cm.sup.2, about
162 cm.sup.2, about 175 cm.sup.2, about 225 cm.sup.2, about 235
cm.sup.2, about 300 cm.sup.2, about 1720 cm.sup.2, about 25
cm.sup.2 to about 75 cm.sup.2, about 25 cm.sup.2 to about 225
cm.sup.2, or about 25 cm.sup.2 to about 1720 cm.sup.2.
[0023] In another aspect, a bag described herein may have a volume
of from about 50 ml to about 100 liters, about 100 ml to about 100
liters, about 150 ml to about 100 liters, about 200 ml to about 100
liters, about 250 ml to about 100 liters, about 500 ml to about 100
liters, about 1 liter to about 100 liters, about 1 liter to about
75 liters, about 1 liter to about 50 liters, about 1 liter to about
25 liters, about 1 liter to about 20 liters, about 1 liter to about
15 liters, about 1 liter to about 10 liters, about 1 liter to about
5 liters, about 1 liter to about 2.5 liters, or about 1 liter to
about 2 liters.
[0024] In yet another aspect, activation described herein may be
carried out in the presence of the T cell activation stimulus.
[0025] In an aspect, cytokines described herein may include
interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 15 (IL-15),
and/or interleukin 21 (IL-21).
[0026] In another aspect, the concentration of IL-7 may be no more
than about 1 ng/ml, no more than about 2 ng/ml, no more than about
3 ng/ml, no more than about 4 ng/ml, no more than about 5 ng/ml, no
more than about 6 ng/ml, no more than about 7 ng/ml, no more than
about 8 ng/ml, no more than about 9 ng/ml, no more than about 10
ng/ml, no more than about 11 ng/ml, no more than about 12 ng/ml, no
more than about 13 ng/ml, no more than about 14 ng/ml, no more than
about 15 ng/ml, no more than about 16 ng/ml, no more than about 17
ng/ml, no more than about 18 ng/ml, no more than about 19 ng/ml, no
more than about 20 ng/ml, no more than about 25 ng/ml, no more than
about 30 ng/ml, no more than about 35 ng/ml, no more than about 40
ng/ml, no more than about 45 ng/ml, no more than about 50 ng/ml, no
more than about 60 ng/ml, no more than about 70 ng/ml, no more than
about 80 ng/ml, no more than about 90 ng/ml, or no more than about
100 ng/ml.
[0027] In another aspect, the concentration of IL-7 may be from
about 1 ng/ml to 100 ng/ml, about 1 ng/ml to 90 ng/ml, about 1
ng/ml to 80 ng/ml, about 1 ng/ml to 70 ng/ml, about 1 ng/ml to 60
ng/ml, about 1 ng/ml to 50 ng/ml, about 1 ng/ml to 40 ng/ml, about
1 ng/ml to 30 ng/ml, about 1 ng/ml to 20 ng/ml, about 1 ng/ml to 15
ng/ml, about 1 ng/ml to 10 ng/ml, about 2 ng/ml to 10 ng/ml, about
4 ng/ml to 10 ng/ml, about 6 ng/ml to 10 ng/ml, or about 5 ng/ml to
10 ng/ml.
[0028] In yet another aspect, the concentration of IL-15 may be no
more than about 5 ng/ml, no more than about 10 ng/ml, no more than
about 15 ng/ml, no more than about 20 ng/ml, no more than about 25
ng/ml, no more than about 30 ng/ml, no more than about 35 ng/ml, no
more than about 40 ng/ml, no more than about 45 ng/ml, no more than
about 50 ng/ml, no more than about 60 ng/ml, no more than about 70
ng/ml, no more than about 80 ng/ml, no more than about 90 ng/ml, no
more than about 100 ng/ml, no more than about 110 ng/ml, no more
than about 120 ng/ml, no more than about 130 ng/ml, no more than
about 140 ng/ml, no more than about 150 ng/ml, 200 ng/ml, 250
ng/ml, 300 ng/ml, 350 ng/ml, 400 ng/ml, 450 ng/ml, or 500
ng/ml.
[0029] In another aspect, the concentration of IL-15 may be from
about 5 ng/ml to 500 ng/ml, about 5 ng/ml to 400 ng/ml, about 5
ng/ml to 300 ng/ml, about 5 ng/ml to 200 ng/ml, about 5 ng/ml to
150 ng/ml, about 5 ng/ml to 100 ng/ml, about 10 ng/ml to 100 ng/ml,
about 20 ng/ml to 100 ng/ml, about 30 ng/ml to 100 ng/ml, about 40
ng/ml to 100 ng/ml, about 50 ng/ml to 100 ng/ml, about 60 ng/ml to
100 ng/ml, about 70 ng/ml to 100 ng/ml, about 80 ng/ml to 100
ng/ml, about 90 ng/ml to 100 ng/ml, about 1 ng/ml to 50 ng/ml,
about 5 ng/ml to 50 ng/ml, about 10 ng/ml to 50 ng/ml, or about 20
ng/ml to 50 ng/ml.
[0030] In another aspect, the concentration of IL-2 may be no more
than about 1000 IU/ml, no more than about 950 IU/ml, no more than
about 900 IU/ml, no more than about 850 IU/ml, no more than about
800 IU/ml, no more than about 750 IU/ml, no more than about 700
IU/ml, no more than about 650 IU/ml, no more than about 600 IU/ml,
no more than about 550 IU/ml, no more than about 500 IU/ml, no more
than about 450 IU/ml, no more than about 400 IU/ml, no more than
about 350 IU/ml, no more than about 300 IU/ml, no more than about
250 IU/ml, no more than about 200 IU/ml, no more than about 150
IU/ml, no more than about 100 IU/ml, no more than about 90 IU/ml,
no more than about 80 IU/ml, no more than about 70 IU/ml, no more
than about 65 IU/ml, no more than about 60 IU/ml, no more than
about 55 IU/ml, no more than about 50 IU/ml, no more than about 40
IU/ml, no more than about 30 IU/ml, no more than about 20 IU/ml, no
more than about 10 IU/ml, or no more than about 5 IU/ml.
[0031] In another aspect, the concentration of IL-2 may be from
about 10 IU/ml to 1000 IU/ml, about 20 IU/ml to 900 IU/ml, about 30
IU/ml to 800 IU/ml, about 40 IU/ml to 700 IU/ml, about 50 IU/ml to
600 IU/ml, about 50 IU/ml to 550 IU/ml, about 50 IU/ml to 500
IU/ml, about 50 IU/ml to 450 IU/ml, about 50 IU/ml to 400 IU/ml,
about 50 IU/ml to 350 IU/ml, about 50 IU/ml to 300 IU/ml, about 50
IU/ml to 250 IU/ml, about 50 IU/ml to 200 IU/ml, about 50 IU/ml to
150 IU/ml, or about 50 IU/ml to 100 IU/ml.
[0032] In another aspect, the concentration of IL-21 may be no more
than about 1 ng/ml, no more than about 2 ng/ml, no more than about
3 ng/ml, no more than about 4 ng/ml, no more than about 5 ng/ml, no
more than about 6 ng/ml, no more than about 7 ng/ml, no more than
about 8 ng/ml, no more than about 9 ng/ml, no more than about 10
ng/ml, no more than about 11 ng/ml, no more than about 12 ng/ml, no
more than about 13 ng/ml, no more than about 14 ng/ml, no more than
about 15 ng/ml, no more than about 16 ng/ml, no more than about 17
ng/ml, no more than about 18 ng/ml, no more than about 19 ng/ml, no
more than about 20 ng/ml, no more than about 25 ng/ml, no more than
about 30 ng/ml, no more than about 35 ng/ml, no more than about 40
ng/ml, no more than about 45 ng/ml, no more than about 50 ng/ml, no
more than about 60 ng/ml, no more than about 70 ng/ml, no more than
about 80 ng/ml, no more than about 90 ng/ml, or no more than about
100 ng/ml.
[0033] In another aspect, the concentration of IL-21 may be from
about 1 ng/ml to 100 ng/ml, about 1 ng/ml to 90 ng/ml, about 1
ng/ml to 80 ng/ml, about 1 ng/ml to 70 ng/ml, about 1 ng/ml to 60
ng/ml, about 1 ng/ml to 50 ng/ml, about 1 ng/ml to 40 ng/ml, about
1 ng/ml to 30 ng/ml, about 1 ng/ml to 20 ng/ml, about 1 ng/ml to 15
ng/ml, about 1 ng/ml to 10 ng/ml, about 2 ng/ml to 10 ng/ml, about
4 ng/ml to 10 ng/ml, about 6 ng/ml to 10 ng/ml, about 5 ng/ml to 10
ng/ml, about 10 ng/ml to 20 ng/ml, about 10 ng/ml to 30 ng/ml,
about 10 ng/ml to 40 ng/ml, about 10 ng/ml to 50 ng/ml, about 10
ng/ml to 60 ng/ml, about 10 ng/ml to 70 ng/ml, about 10 ng/ml to 80
ng/ml, about 10 ng/ml to 90 ng/ml, or about 10 ng/ml to 100
ng/ml.
[0034] In an aspect, transducing described herein may be carried
out within a period of no more than about 1 hour, no more than
about 2 hours, no more than about 3 hours, no more than about 4
hours, no more than about 5 hours, no more than about 6 hours, no
more than about 7 hours, no more than about 8 hours, no more than
about 9 hours, no more than about 10 hours, no more than about 11
hours, no more than about 12 hours, no more than about 14 hours, no
more than about 16 hours, no more than about 18 hours, no more than
about 20 hours, no more than about 22 hours, no more than about 24
hours, no more than about 26 hours, no more than about 28 hours, no
more than about 30 hours, no more than about 36 hours, no more than
about 42 hours, no more than about 48 hours, no more than about 54
hours, no more than about 60 hours, no more than about 66 hours, no
more than about 72 hours, no more than about 84 hours, no more than
about 96 hours, no more than about 108 hours, or no more than about
120 hours.
[0035] In yet another aspect, transducing described herein may be
carried out within a period of from about 1 hour to about 120
hours, about 1 hour to about 108 hours, about 1 hour to about 96
hours, about 1 hour to about 72 hours, about 1 hour to about 48
hours, about 1 hour to about 36 hours, about 1 hour to about 24
hours, about 1 hour to about 12 hours, about 2 hours to about 24
hours, about 4 hours to about 24 hours, about 12 hours to about 24
hours, about 12 hours to about 48 hours, about 12 hour to about 72
hours, about 24 hours to about 72 hours, or about 36 hours to about
72 hours.
[0036] In another aspect, viral vector described herein may be a
.gamma.-retroviral vector expressing a T cell receptor (TCR).
[0037] In yet another aspect, viral vector described herein may be
a lentiviral vector expressing a TCR.
[0038] In an aspect, transducing described herein may be carried
out in the presence of the T cell activation stimulus.
[0039] In an aspect, expanding described herein may be carried out
in the presence of the T cell activation stimulus.
[0040] In an aspect, expanding described herein may be carried out
within a period of no more than about 1 day, no more than about 2
days, no more than about 3 days, no more than about 4 days, no more
than about 5 days, no more than about 6 days, no more than about 7
days, no more than about 8 days, no more than about 9 days, no more
than about 10 days, no more than about 15 days, no more than about
20 days, no more than about 25 days, or no more than about 30
days.
[0041] In another aspect, expanding described herein may be carried
out within a period of from about 1 day to about 30 days, about 1
day to about 25 days, about 1 day to about 20 days, about 1 day to
about 15 days, about 1 day to about 10 days, about 2 days to about
10 days, about 3 days to about 10 days, about 4 days to about 10
days, about 4 days to about 30 days, about 6 days to about 25 days,
about 10 days to about 30 days, or about 12 days to about 30
days.
[0042] In an aspect, the number of the obtained T cells may be at
least about 1.times.10.sup.9, may be at least about
2.times.10.sup.9, may be at least about 3.times.10.sup.9, may be at
least about 4.times.10.sup.9, may be at least about
5.times.10.sup.9, may be at least about 6.times.10.sup.9, may be at
least about 7.times.10.sup.9, may be at least about
8.times.10.sup.9, may be at least about 9.times.10.sup.9, may be at
least about 1.times.10.sup.10, may be at least about
5.times.10.sup.10, may be at least about 1.times.10.sup.11, may be
at least about 5.times.10.sup.11, may be at least about
1.times.10.sup.12, may be at least about 5.times.10.sup.12 or may
be at least about 1.times.10.sup.13 cells.
[0043] In another aspect, the number of the obtained T cells may be
from about 1.times.10.sup.9 to about 1.times.10.sup.13, about
1.times.10.sup.9 to about 5.times.10.sup.12, about 1.times.10.sup.9
to about 1.times.10.sup.12, about 1.times.10.sup.9 to about
5.times.10.sup.11, about 1.times.10.sup.9 to about
1.times.10.sup.11, about 1.times.10.sup.9 to about
5.times.10.sup.10, about 1.times.10.sup.9 to about
1.times.10.sup.10, about 2.times.10.sup.9 to about
1.times.10.sup.10, about 3.times.10.sup.9 to about
1.times.10.sup.10, about 4.times.10.sup.9 to about
1.times.10.sup.10, about 5.times.10.sup.9 to about
1.times.10.sup.10, about 6.times.10.sup.9 to about
1.times.10.sup.10, about 7.times.10.sup.9 to about
1.times.10.sup.10, about 8.times.10.sup.9 to about
1.times.10.sup.10, or about 9.times.10.sup.9 to about
1.times.10.sup.10 cells.
[0044] In an aspect, the obtained T cells may be a CD3.sup.+
CD8.sup.+ T cell and/or CD3.sup.+ CD4+ T cells.
[0045] In another aspect, PBMC may be obtained from the
patient.
[0046] In yet another aspect, the present disclosure relates to
genetically transduced T cells produced by the method described
herein.
[0047] In another aspect, the present disclosure relates to
pharmaceutical compositions containing the genetically transduced T
cells produced by the method described herein and pharmaceutically
acceptable carriers.
[0048] In another aspect, the present disclosure relates to a
method of preparing a T cell population, including thawing frozen
peripheral blood mononuclear cells (PBMC), resting the thawed PBMC,
activating the T cell in the rested PBMC with an anti-CD3 antibody
and an anti-CD28 antibody immobilized on a solid phase, expanding
the activated T cell, and obtaining the T cell population
comprising the expanded T cell.
[0049] In yet another aspect, the present disclosure relates to a T
cell population prepared by the method described herein.
[0050] In another aspect, the present disclosure relates to methods
of treating a patient or individual having a cancer or in need of a
treatment thereof, comprising administering to the patient an
effective amount of the expanded T cells described herein. In an
aspect, the patient or individual in need thereof is a cancer
patient. In an aspect, the cancer to be treated is selected from
one or more of hepatocellular carcinoma (HCC), colorectal carcinoma
(CRC), glioblastoma (GB), gastric cancer (GC), esophageal cancer,
non-small cell lung cancer (NSCLC), pancreatic cancer (PC), renal
cell carcinoma (RCC), benign prostate hyperplasia (BPH), prostate
cancer (PCA), ovarian cancer (OC), melanoma, breast cancer, chronic
lymphocytic leukemia (CLL), Merkel cell carcinoma (MCC), small cell
lung cancer (SCLC), Non-Hodgkin lymphoma (NHL), acute myeloid
leukemia (AML), gallbladder cancer and cholangiocarcinoma (GBC,
CCC), urinary bladder cancer (UBC), acute lymphocytic leukemia
(ALL), and uterine cancer (UEC).
[0051] In another aspect, the expanding may be carried out in the
presence of at least one cytokine selected from the group
consisting of IL-2, IL-7, IL-12, IL-15, and IL-21. In an aspect,
the expansion takes place in the presence of a combination IL-7 and
IL-15.
[0052] In another aspect, the thawing, the resting, the activating,
the transducing, the expanding, and/or the obtaining may be
performed in a closed system.
[0053] In another aspect, the present disclosure relates to a
method of preparing a T cell population, including obtaining fresh
peripheral blood mononuclear cells (PBMC), i.e., PBMC is not
obtained by thawing cryopreserved PBMC, activating the T cell in
the fresh PBMC with an anti-CD3 antibody and an anti-CD28 antibody,
transducing the activated T cell with a viral vector, expanding the
transduced T cell, and harvesting the expanded T cell.
[0054] In an aspect, the obtaining and the activating may be
performed for no more than 1 day.
[0055] In an aspect, the expanding may be performed for more than 1
day.
[0056] In another aspect, the expanding may be performed for from
about 1 day to 2 days, from about 1 day to 3 days, from about 1 day
to about 4 days, from about 1 day to about 5 days, from about 1 day
to 6 days, from about 1 day to 7 days, from about 1 day to 8 days,
from about 1 day to 9 days, from about 1 day to 10 days, from about
2 days to 3 days, from about 2 days to 4 days, from about 2 days to
5 days, from about 2 days to 6 days, from about 2 days to 7 days,
from about 2 days to 8 days, from about 2 days to 9 days, from
about 2 days to 10 days, from about 3 days to 4 days, from about 3
days to 5 days, from about 3 days to 6 days, from about 3 days to 7
days, from about 3 days to 8 days, from about 3 days to 9 days,
from about 3 days to 10 days, from about 4 days to 5 days, from
about 4 days to 6 days, from about 4 days to 7 days, from about 4
days to 8 days, from about 4 days to 9 days, from about 4 days to
10 days, from about 5 days to 6 days, from about 5 days to 7 days,
from about 5 days to 8 days, from about 5 days to 9 days, or from
about 5 days to 10 days.
[0057] In another aspect, the harvesting may be performed after the
activating within from about 4 days to about 12 days, from about 4
days to about 11 days, from about 4 days to about 10 days, from
about 4 days to about 9 days, from about 4 days to about 8 days,
from about 4 days to about 7 days, from about 4 days to about 6
days, from about 4 days to about 5 days, from about 5 days to about
12 days, from about 5 days to about 11 days, from about 5 days to
about 10 days, from about 5 days to about 9 days, from about 5 days
to about 8 days, from about 5 days to about 7 days, or from about 5
days to about 6 days.
[0058] In another aspect, the number of the harvested T cells may
be selected from the group consisting of from about
2.times.10.sup.9 to about 5.times.10.sup.9, about 5.times.10.sup.9
to about 10.times.10.sup.9, about 10.times.10.sup.9 to about
15.times.10.sup.9, about 5.times.10.sup.9 to about
35.times.10.sup.9, about 5.times.10.sup.9 to about
30.times.10.sup.9, about 10.times.10.sup.9 to about
30.times.10.sup.9, about 15.times.10.sup.9 to about
20.times.10.sup.9, about 20.times.10.sup.9 to about
35.times.10.sup.9, about 24.times.10.sup.9 to about
33.times.10.sup.9, and about 24.8.times.10.sup.9 to about
32.2.times.10.sup.9.
[0059] In another aspect, the activating, the transducing, the
expanding, and the harvesting may be performed in a closed or
semi-closed system.
[0060] In another aspect, the closed system may be CliniMACS,
Prodigy.TM., WAVE (XURI.TM.) Bioreactor, WAVE (XURI.TM.) Bioreactor
in combination with BioSafe Sepax.TM. II, G-Rex/GatheRex.TM. closed
system, or G-Rex/GatheRex.TM. closed system in combination with
BioSafe Sepax.TM. II.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] For a further understanding of the nature, objects, and
advantages of the present disclosure, reference should be had to
the following detailed description, read in conjunction with the
following drawings, wherein like reference numerals denote like
elements.
[0062] FIGS. 1A and 1B show loss of T.sub.naive/scm and T.sub.cm
phenotype by prolonging ex vivo culturing of T cells obtained from
different donors.
[0063] FIG. 2 shows reduction of IFN-.gamma. secretion in cells
grown on Day 15 as compared with that grown on Day 10 from
different donors.
[0064] FIG. 3 shows an experimental design to test the effect of
resting conditions on T cell activation and expansion.
[0065] FIG. 4 shows CD25, CD69, and hLDL-R expression levels in
different experimental groups.
[0066] FIGS. 5A and 5B show fold expansion and cell viability in
different experimental groups on Day 7 expansion and Day 10
expansion, respectively.
[0067] FIG. 6 shows fold expansion and viability of activated T
cells transduced with a viral vector in different experimental
groups on Day 9.
[0068] FIG. 7 shows fold expansion and viability of activated T
cells transduced with a viral vector in different experimental
groups on Day 9.
[0069] FIG. 8 shows transgene expression in T cells resulting from
different resting time and in different scale production.
[0070] FIG. 9 shows fold expansion on Day 10 resulting from
different resting time and in different scale production.
[0071] FIG. 10 shows an experimental design to test the effect of
concentration of anti-CD3 and anti-CD28 antibodies on T cell
activation.
[0072] FIG. 11 shows CD25, CD69, and hLDL-R expression in T cells
activated by different concentrations of anti-CD3 and anti-CD28
antibodies.
[0073] FIG. 12 shows, on Day 10 expansion, cell counts of T cells
activated by different concentrations of anti-CD3 and anti-CD28
antibodies in the presence of different concentrations of
IL-15.
[0074] FIG. 13 shows tetramer staining of recombinant
TCR-transduced T cells activated by different concentrations of
anti-CD3 and anti-CD28 antibodies in the presence of different
concentrations of IL-15.
[0075] FIG. 14A shows the percentage of
CD3.sup.+CD8.sup.+Tetramer.sup.+ T cells resulting from different
durations of activation.
[0076] FIG. 14B shows transgene expression resulting from different
durations of activation.
[0077] FIG. 15 shows CD25, CD69, and LDL-R expression in T cells
activated by plate-bound or flask-bound anti-CD3 and anti-CD28
antibodies.
[0078] FIG. 16A shows levels of transduction in flask-bound (FB)
and plate-bound (PB) activated T cells.
[0079] FIG. 16B shows fold expansion in flask-bound (FB) and
plate-bound (PB) activated T cells.
[0080] FIG. 17 shows antigen specific IFN-.gamma. levels elicited
by flask-bound (FB) activated LV-R73 (a lentiviral vector
expressing a T cell receptor) transduced T cells and plate-bound
(PB) activated transduced T cells in response to tumor cells
expressing a tumor associated antigen (TAA) in different
donors.
[0081] FIG. 18 shows an experimental design to test the effect of
using bags and plates coated with anti-CD3 and anti-CD28 antibodies
on T cell activation.
[0082] FIG. 19 shows CD25, CD69, and LDL-R expression in T cells
activated in bag-bound or flask-bound anti-CD3 and anti-CD28
antibodies.
[0083] FIG. 20 shows, on Day 6 expansion, cell expansion resulting
from T cells activated by bag-bound antibodies at different
concentrations and that of T cells activated under FB
conditions.
[0084] FIG. 21 shows, on Day 10 expansion, cell expansion resulting
from T cells activated by bag-bound antibodies at different
concentrations and that of T cells activated under FB
conditions.
[0085] FIG. 22 shows a T cell manufacturing process according to
one embodiment of the present disclosure.
[0086] FIG. 23A shows fold expansion of T cells manufactured
according to one embodiment of the present disclosure.
[0087] FIG. 23B shows transduced TCR expression of T cells
manufactured according to one embodiment of the present
disclosure.
[0088] FIG. 23C shows phenotypes of T cells manufactured according
to one embodiment of the present disclosure.
[0089] FIG. 23D shows tumor cell growth inhibitory activity of T
cells manufactured according to one embodiment of the present
disclosure.
[0090] FIG. 23E shows tumor cell growth inhibitory activity of T
cells manufactured according to another embodiment of the present
disclosure.
[0091] FIG. 23F shows tumor cell growth inhibitory activity of T
cells manufactured according to another embodiment of the present
disclosure.
[0092] FIG. 23G shows tumor cell killing activity of T cells
manufactured according to another embodiment of the present
disclosure.
[0093] FIG. 23H shows tumor cell killing activity of T cells
manufactured according to another embodiment of the present
disclosure.
[0094] FIG. 24 shows T cell manufacturing process with overnight
rest (about 16 hours).
[0095] FIG. 25A shows fold expansion of T cells manufactured with
overnight rest (about 16 hours).
[0096] FIG. 25B shows transduced TCR expression of T cells
manufactured with overnight rest (about 16 hours).
[0097] FIG. 25C shows phenotypes of T cells manufactured with
overnight rest (about 16 hours).
[0098] FIG. 25D shows tumor cell growth inhibitory activity of T
cells manufactured with overnight rest (about 16 hours).
[0099] FIGS. 25E and 25F show cytotoxic activity of T cells
manufactured with overnight rest (about 16 hours).
[0100] FIG. 26 shows ex vivo manipulation protocol in open and
closed systems.
[0101] FIG. 27 shows ex vivo manipulation protocol in closed system
in accordance with one embodiment of the present disclosure.
[0102] FIG. 28 shows ex vivo manipulation protocol in closed system
in accordance with another embodiment of the present
disclosure.
[0103] FIG. 29 shows IFN-.gamma. release from T cells manufactured
in open and closed systems.
[0104] FIG. 30 shows a schematic of T cell manufacturing in
accordance with some embodiments of the present disclosure.
[0105] FIG. 31 shows a representative turnaround time from
leukapheresis collection to infusion-ready in accordance with one
embodiment of the present disclosure. LP.sup.#:Leukapheresis
collection, processing & freeze (optional). CoA: Additional
time required for issuance of Certificate of Analysis.
[0106] FIG. 32 shows a T cell manufacturing process in accordance
with one embodiment of the present disclosure.
[0107] FIG. 33 shows T cell memory phenotyping of T cells produced
by a manufacturing process in accordance with one embodiment of the
present disclosure.
[0108] FIG. 34 shows CD27 and CD28 co-stimulation phenotyping of T
cells produced by a manufacturing process in accordance with one
embodiment of the present disclosure.
[0109] FIG. 35 shows T cell growth induced by IL-7, IL-15, or IL-2
decreases in an expansion time-dependent manner in accordance with
one embodiment of the present disclosure.
[0110] FIG. 36 shows IFN-.gamma. secretion decreases in an
expansion time-dependent manner in accordance with one embodiment
of the present disclosure.
[0111] FIG. 37 shows EC.sub.50 increases in an expansion
time-dependent manner in accordance with one embodiment of the
present disclosure.
[0112] FIG. 38 shows expansion metrics in accordance with one
embodiment of the present disclosure.
[0113] FIG. 39 shows surface expression of TCR in accordance with
one embodiment of the present disclosure.
[0114] FIG. 40 shows T-cell memory phenotype of the final products
in accordance with one embodiment of the present disclosure.
[0115] FIG. 41 shows IFN-.gamma. release in response to exposure to
target cells in accordance with one embodiment of the present
disclosure.
[0116] FIG. 42 shows EC.sub.50 determination in accordance with one
embodiment of the present disclosure.
[0117] FIG. 43 shows cytotoxic potential of T cells in accordance
with one embodiment of the present disclosure.
[0118] FIG. 44 shows a comparison in cell recovery between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0119] FIG. 45 shows a comparison in cell viability between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0120] FIG. 46 shows a comparison in fold expansion between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0121] FIG. 47 shows a comparison in cell phenotype between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0122] FIG. 48 shows a comparison in cell phenotype between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0123] FIG. 49 shows TCR expression of T cell products in
accordance with an embodiment of the present disclosure.
[0124] FIG. 50 shows a comparison in TCR expression between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0125] FIG. 51 shows a comparison in TCR expression between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0126] FIG. 52 shows gating scheme and T.sub.memory subsets in
accordance with an embodiment of the present disclosure.
[0127] FIG. 53 shows a comparison in cell phenotype between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0128] FIG. 54 shows cytokine expression in T cell products in
accordance with an embodiment of the present disclosure.
[0129] FIG. 55 shows cytokine expression in T cell products
obtained from healthy donor in accordance with an embodiment of the
present disclosure.
[0130] FIG. 56 shows a comparison in cytokine expression between T
cell products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0131] FIG. 57 shows IFN-.gamma. release from T cell products
obtained from cancer patients in accordance with an embodiment of
the present disclosure.
[0132] FIG. 58 shows IFN-.gamma. release from T cell products
obtained from healthy donors in accordance with an embodiment of
the present disclosure.
[0133] FIG. 59 shows IFN-.gamma. release from T cell products
obtained from healthy donors in accordance with an embodiment of
the present disclosure.
[0134] FIG. 60 shows IFN-.gamma. release from T cell products
obtained from cancer patients in accordance with an embodiment of
the present disclosure.
[0135] FIG. 61 shows cell killing activity of T cell products
obtained from healthy donors in accordance with an embodiment of
the present disclosure.
[0136] FIG. 62 shows cell killing activity of T cell products
obtained from healthy donors in accordance with an embodiment of
the present disclosure.
[0137] FIG. 63A shows a comparison in cell killing between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0138] FIG. 63B shows a comparison in cell killing between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
[0139] FIG. 63C shows a comparison in cell killing between T cell
products obtained from healthy donors and cancer patients in
accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION
[0140] In an aspect, the disclosure provides for T cells
populations produced by a method including thawing frozen
peripheral blood mononuclear cells (PBMC), resting the thawed PBMC,
activating the T cell in the rested PBMC with an anti-CD3 antibody
and an anti-CD28 antibody immobilized on a solid phase, expanding
the activated T cell, and obtaining the T cell population
comprising the expanded T cell.
[0141] In an aspect, the disclosure provides for methods of
transducing a T cell including thawing frozen peripheral blood
mononuclear cells (PBMC), resting the thawed PBMC, activating the T
cell in the cultured PBMC with an anti-CD3 antibody and an
anti-CD28 antibody, transducing the activated T cell with a viral
vector, expanding the transduced T cell, and obtaining the expanded
T cells; method of preparing a T cell population, including thawing
frozen peripheral blood mononuclear cells (PBMC), resting the
thawed PBMC, activating the T cell in the rested PBMC with an
anti-CD3 antibody and an anti-CD28 antibody immobilized on a solid
phase, expanding the activated T cell, and obtaining the T cell
population comprising the expanded T cell; and methods of treating
a patient or individual having a cancer or in need of a treatment
thereof, comprising administering to the patient an effective
amount of the expanded T cells described herein. In an aspect, the
patient or individual in need thereof is a cancer patient. In an
aspect, the cancer to be treated is selected from one or more of
hepatocellular carcinoma (HCC), colorectal carcinoma (CRC),
glioblastoma (GB), gastric cancer (GC), esophageal cancer,
non-small cell lung cancer (NSCLC), pancreatic cancer (PC), renal
cell carcinoma (RCC), benign prostate hyperplasia (BPH), prostate
cancer (PCA), ovarian cancer (OC), melanoma, breast cancer, chronic
lymphocytic leukemia (CLL), Merkel cell carcinoma (MCC), small cell
lung cancer (SCLC), Non-Hodgkin lymphoma (NHL), acute myeloid
leukemia (AML), gallbladder cancer and cholangiocarcinoma (GBC,
CCC), urinary bladder cancer (UBC), acute lymphocytic leukemia
(ALL), and uterine cancer (UEC).
[0142] T-cell based immunotherapy targets peptide epitopes derived
from tumor-associated or tumor-specific proteins, which are
presented by molecules of the major histocompatibility complex
(MHC). The antigens that are recognized by the tumor specific T
lymphocytes, that is, the epitopes thereof, can be molecules
derived from all protein classes, such as enzymes, receptors,
transcription factors, etc. which are expressed and, as compared to
unaltered cells of the same origin, usually up-regulated in cells
of the respective tumor.
[0143] There are two classes of MHC-molecules, MHC class I and MHC
class II. MHC class I molecules are composed of an alpha heavy
chain and beta-2-microglobulin, MHC class II molecules of an alpha
and a beta chain. Their three-dimensional conformation results in a
binding groove, which is used for non-covalent interaction with
peptides. MHC class I molecules can be found on most nucleated
cells. They present peptides that result from proteolytic cleavage
of predominantly endogenous proteins, defective ribosomal products
(DRIPs) and larger peptides. However, peptides derived from
endosomal compartments or exogenous sources are also frequently
found on MHC class I molecules. This non-classical way of class I
presentation is referred to as cross-presentation. MHC class II
molecules can be found predominantly on professional antigen
presenting cells (APCs), and primarily present peptides of
exogenous or transmembrane proteins that are taken up by APCs e.g.,
during endocytosis, and are subsequently processed.
[0144] Complexes of peptide and MHC class I are recognized by
CD8-positive T-cells bearing the appropriate T-cell receptor (TCR),
whereas complexes of peptide and MHC class II molecules are
recognized by CD4-positive-helper-T-cells bearing the appropriate
TCR. It is well known that the TCR, the peptide and the MHC are
thereby present in a stoichiometric amount of 1:1:1.
[0145] CD4-positive helper T-cells play an important role in
inducing and sustaining effective responses by CD8-positive
cytotoxic T-cells. The identification of CD4-positive T-cell
epitopes derived from tumor associated antigens (TAA) is of great
importance for the development of pharmaceutical products for
triggering anti-tumor immune responses. At the tumor site, T helper
cells, support a cytotoxic T-cell-(CTL-) friendly cytokine milieu
and attract effector cells, e.g., CTLs, natural killer (NK) cells,
macrophages, and granulocytes.
[0146] In the absence of inflammation, expression of MHC class II
molecules is mainly restricted to cells of the immune system,
especially professional antigen-presenting cells (APC), e.g.,
monocytes, monocyte-derived cells, macrophages, dendritic cells. In
cancer patients, cells of the tumor have been found to express MHC
class II molecules. Elongated (longer) peptides of the description
can function as MHC class II active epitopes.
[0147] T-helper cells, activated by MHC class II epitopes, play an
important role in orchestrating the effector function of CTLs in
anti-tumor immunity. T-helper cell epitopes that trigger a T-helper
cell response of the TH1 type support effector functions of
CD8-positive killer T-cells, which include cytotoxic functions
directed against tumor cells displaying tumor-associated
peptide/MHC complexes on their cell surfaces. In this way
tumor-associated T-helper cell peptide epitopes, alone or in
combination with other tumor-associated peptides, can serve as
active pharmaceutical ingredients of vaccine compositions that
stimulate anti-tumor immune responses.
[0148] It was shown in mammalian animal models, e.g., mice, that
even in the absence of CD8-positive T lymphocytes, CD4-positive
T-cells are sufficient for inhibiting manifestation of tumors via
inhibition of angiogenesis by secretion of interferon-gamma
(IFN-.gamma.). There is evidence for CD4-positive T-cells as direct
anti-tumor effectors.
[0149] Since the constitutive expression of HLA class II molecules
is usually limited to immune cells, the possibility of isolating
class II peptides directly from primary tumors was previously not
considered possible. However, Dengjel et al. were successful in
identifying a number of MHC Class II epitopes directly from tumors
(WO 2007/028574, EP 1 760 088 B1,the contents of which are herein
incorporated by reference in their entirety).
[0150] Since both types of response, CD8 and CD4 dependent,
contribute jointly and synergistically to the anti-tumor effect,
the identification and characterization of tumor-associated
antigens recognized by either CD8+ T-cells (ligand: MHC class I
molecule+peptide epitope) or by CD4-positive T-helper cells
(ligand: MHC class II molecule+peptide epitope) is important in the
development of tumor vaccines.
[0151] For an MHC class I peptide to trigger (elicit) a cellular
immune response, it also must bind to an MHC-molecule. This process
is dependent on the allele of the MHC-molecule and specific
polymorphisms of the amino acid sequence of the peptide.
MHC-class-1-binding peptides are usually 8-12 amino acid residues
in length and usually contain two conserved residues ("anchors") in
their sequence that interact with the corresponding binding groove
of the MHC-molecule. In this way, each MHC allele has a "binding
motif" determining which peptides can bind specifically to the
binding groove.
[0152] In the MHC class I dependent immune reaction, peptides not
only have to be able to bind to certain MHC class I molecules
expressed by tumor cells, they subsequently also have to be
recognized by T-cells bearing specific T-cell receptors (TCR).
[0153] For proteins to be recognized by T-lymphocytes as
tumor-specific or -associated antigens, and to be used in a
therapy, particular prerequisites must be fulfilled. The antigen
should be expressed mainly by tumor cells and not, or in comparably
small amounts, by normal healthy tissues. In a preferred
embodiment, the peptide should be over-presented by tumor cells as
compared to normal healthy tissues. It is furthermore desirable
that the respective antigen is not only present in a type of tumor,
but also in high concentrations (i.e., copy numbers of the
respective peptide per cell). Tumor-specific and tumor-associated
antigens are often derived from proteins directly involved in
transformation of a normal cell to a tumor cell due to their
function, e.g., in cell cycle control or suppression of apoptosis.
Additionally, downstream targets of the proteins directly causative
for a transformation may be up-regulated and thus may be indirectly
tumor-associated. Such indirect tumor-associated antigens may also
be targets of a vaccination approach. Epitopes are present in the
amino acid sequence of the antigen, in order to ensure that such a
peptide ("immunogenic peptide"), being derived from a tumor
associated antigen, and leads to an in vitro or in vivo
T-cell-response.
[0154] Therefore, TAAs are a starting point for the development of
a T-cell based therapy including but not limited to tumor vaccines.
The methods for identifying and characterizing the TAAs are usually
based on the use of T-cells that can be isolated from patients or
healthy subjects, or they are based on the generation of
differential transcription profiles or differential peptide
expression patterns between tumors and normal tissues. However, the
identification of genes over-expressed in tumor tissues or human
tumor cell lines, or selectively expressed in such tissues or cell
lines, does not provide precise information as to the use of the
antigens being transcribed from these genes in an immune therapy.
This is because only an individual subpopulation of epitopes of
these antigens are suitable for such an application since a T-cell
with a corresponding TCR has to be present and the immunological
tolerance for this particular epitope needs to be absent or
minimal. In a very preferred embodiment of the description it is
therefore important to select only those over- or selectively
presented peptides against which a functional and/or a
proliferating T-cell can be found. Such a functional T-cell is
defined as a T-cell, which upon stimulation with a specific antigen
can be clonally expanded and is able to execute effector functions
("effector T-cell").
[0155] The term "T-cell receptor (TCR)" as used herein refers to a
protein receptor on T cells that is composed of a heterodimer of an
alpha (.alpha.) and beta (.beta.) chain, although in some cells the
TCR consists of gamma and delta (.gamma./.delta.) chains. In
embodiments of the disclosure, the TCR may be modified on any cell
comprising a TCR, including a helper T cell, a cytotoxic T cell, a
memory T cell, regulatory T cell, natural killer T cell, and gamma
delta T cell, for example.
[0156] TCR is a molecule found on the surface of T lymphocytes (or
T cells) that is generally responsible for recognizing antigens
bound to major histocompatibility complex (MHC) molecules. It is a
heterodimer consisting of an alpha and beta chain in 95% of T
cells, while 5% of T cells have TCRs consisting of gamma and delta
chains. Engagement of the TCR with antigen and MHC results in
activation of its T lymphocyte through a series of biochemical
events mediated by associated enzymes, co-receptors, and
specialized accessory molecules. In immunology, the CD3 antigen (CD
stands for cluster of differentiation) is a protein complex
composed of four distinct chains (CD3-.gamma., CD3.delta., and two
times CD3.epsilon.) in mammals, that associate with molecules known
as the T-cell receptor (TCR) and the .zeta.-chain to generate an
activation signal in T lymphocytes. The TCR, .zeta.-chain, and CD3
molecules together comprise the TCR complex. The CD3-.gamma.,
CD3.delta., and CD3.epsilon. chains are highly related cell surface
proteins of the immunoglobulin superfamily containing a single
extracellular immunoglobulin domain. The transmembrane region of
the CD3 chains is negatively charged, a characteristic that allows
these chains to associate with the positively charged TCR chains
(TCR.alpha. and TCR.beta.). The intracellular tails of the CD3
molecules contain a single conserved motif known as an
immunoreceptor tyrosine-based activation motif or ITAM for short,
which is essential for the signalling capacity of the TCR.
[0157] CD28 is one of the molecules expressed on T cells that
provide co-stimulatory signals, which are required for T cell
activation. CD28 is the receptor for B7.1 (CD80) and B7.2 (CD86).
When activated by Toll-like receptor ligands, the B7.1 expression
is upregulated in antigen presenting cells (APCs). The B7.2
expression on antigen presenting cells is constitutive. CD28 is the
only B7 receptor constitutively expressed on naive T cells.
Stimulation through CD28 in addition to the TCR can provide a
potent co-stimulatory signal to T cells for the production of
various interleukins (IL-2 and IL-6 in particular).
[0158] In an aspect, expansion and/or activation of T cells take
place in the presence of one or more of IL-2, IL-7, IL-10, IL-12,
IL-15, IL-21. In another aspect, expansion and/or activation of T
cells takes place with IL-2 alone, IL-7 alone, IL-15 alone, a
combination of IL-2 and IL-15, or a combination of IL-7 and
IL-15.
[0159] TCR constructs of the present disclosure may be applicable
in subjects having or suspected of having cancer by reducing the
size of a tumor or preventing the growth or re-growth of a tumor in
these subjects. Accordingly, the present disclosure further relates
to a method for reducing growth or preventing tumor formation in a
subject by introducing a TCR construct of the present disclosure
into an isolated T cell of the subject and reintroducing into the
subject the transformed T cell, thereby effecting anti-tumor
responses to reduce or eliminate tumors in the subject. Suitable T
cells that can be used include cytotoxic lymphocytes (CTL) or any
cell having a T cell receptor in need of disruption. As is
well-known to one of skill in the art, various methods are readily
available for isolating these cells from a subject. For example,
using cell surface marker expression or using commercially
available kits (e.g., ISOCELL.TM. from Pierce, Rockford, Ill.).
[0160] It is contemplated that the TCR construct can be introduced
into the subject's own T cells as naked DNA or in a suitable
vector. Methods of stably transfecting T cells by electroporation
using naked DNA in the art. See, e.g., U.S. Pat. No. 6,410,319, the
content of which is incorporated by reference in its entirety.
Naked DNA generally refers to the DNA encoding a TCR of the present
disclosure contained in a plasmid expression vector in proper
orientation for expression. Advantageously, the use of naked DNA
reduces the time required to produce T cells expressing the TCR of
the present disclosure.
[0161] Alternatively, a viral vector (e.g., a retroviral vector,
adenoviral vector, adeno-associated viral vector, or lentiviral
vector) can be used to introduce the TCR construct into T cells.
Suitable vectors for use in accordance with the method of the
present disclosure are non-replicating in the subject's T cells. A
large number of vectors are known that are based on viruses, where
the copy number of the virus maintained in the cell is low enough
to maintain the viability of the cell. Illustrative vectors include
the pFB-neo vectors (STRATAGENE.RTM.) as well as vectors based on
HIV, SV40, EBV, HSV, or BPV.
[0162] Once it is established that the transfected or transduced T
cell is capable of expressing the TCR construct as a surface
membrane protein with the desired regulation and at a desired
level, it can be determined whether the TCR is functional in the
host cell to provide for the desired signal induction.
Subsequently, the transduced T cells are reintroduced or
administered to the subject to activate anti-tumor responses in the
subject.
[0163] To facilitate administration, the transduced T cells
according to the disclosure can be made into a pharmaceutical
composition or made into an implant appropriate for administration
in vivo, with appropriate carriers or diluents, which further can
be pharmaceutically acceptable. The means of making such a
composition or an implant have been described in the art (see, for
instance, Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed.
(1980, the content which is herein incorporated by reference in its
entirety)). Where appropriate, the transduced T cells can be
formulated into a preparation in semisolid or liquid form, such as
a capsule, solution, injection, inhalant, or aerosol, in the usual
ways for their respective route of administration. Means known in
the art can be utilized to prevent or minimize release and
absorption of the composition until it reaches the target tissue or
organ, or to ensure timed-release of the composition. Desirably,
however, a pharmaceutically acceptable form is employed that does
not hinder the cells from expressing the TCR. Thus, desirably the
transduced T cells can be made into a pharmaceutical composition
containing a balanced salt solution, preferably Hanks' balanced
salt solution, or normal saline.
[0164] In certain aspects, the invention includes a method of
making and/or expanding the antigen-specific redirected T cells
that comprises transfecting T cells with an expression vector
containing a DNA construct encoding TCR, then, optionally,
stimulating the cells with antigen positive cells, recombinant
antigen, or an antibody to the receptor to cause the cells to
proliferate.
[0165] In another aspect, a method is provided of stably
transfecting and re-directing T cells by electroporation, or other
non-viral gene transfer (such as, but not limited to sonoporation)
using naked DNA. Most investigators have used viral vectors to
carry heterologous genes into T cells. By using naked DNA, the time
required to produce redirected T cells can be reduced. "Naked DNA"
means DNA encoding a TCR contained in an expression cassette or
vector in proper orientation for expression. The electroporation
method of this disclosure produces stable transfectants that
express and carry on their surfaces the TCR.
[0166] In certain aspects, the T cells are primary human T cells,
such as T cells derived from human peripheral blood mononuclear
cells (PBMC), PBMC collected after stimulation with G-CSF, bone
marrow, or umbilical cord blood. Conditions include the use of mRNA
and DNA and electroporation. Following transfection, cells may be
immediately infused or may be stored. In certain aspects, following
transfection, the cells may be propagated for days, weeks, or
months ex vivo as a bulk population within about 1, 2, 3, 4, 5 days
or more following gene transfer into cells. In a further aspect,
following transfection, the transfectants are cloned and a clone
demonstrating presence of a single integrated or episomally
maintained expression cassette or plasmid, and expression of the
TCR is expanded ex vivo. The clone selected for expansion
demonstrates the capacity to specifically recognize and lyse
peptide-expressing target cells. The recombinant T cells may be
expanded by stimulation with IL-2, or other cytokines that bind the
common gamma-chain (e.g., IL-7, IL-12, IL-15, IL-21, and others).
The recombinant T cells may be expanded by stimulation with
artificial antigen presenting cells. The recombinant T cells may be
expanded on artificial antigen presenting cell or with an antibody,
such as OKT3, which cross links CD3 on the T cell surface. Subsets
of the recombinant T cells may be deleted on artificial antigen
presenting cell or with an antibody, such as Campath, which binds
CD52 on the T cell surface. In a further aspect, the genetically
modified cells may be cryopreserved.
[0167] A composition of the present invention can be provided in
unit dosage form wherein each dosage unit, e.g., an injection,
contains a predetermined amount of the composition, alone or in
appropriate combination with other active agents. The term unit
dosage form as used herein refers to physically discrete units
suitable as unitary dosages for human and animal subjects, each
unit containing a predetermined quantity of the composition of the
present invention, alone or in combination with other active
agents, calculated in an amount sufficient to produce the desired
effect, in association with a pharmaceutically acceptable diluent,
carrier, or vehicle, where appropriate. The specifications for the
novel unit dosage forms of the present invention depend on the
particular pharmacodynamics associated with the pharmaceutical
composition in the particular subject.
[0168] Desirably an effective amount or sufficient number of the
isolated transduced T cells is present in the composition and
introduced into the subject such that long-term, specific,
anti-tumor responses are established to reduce the size of a tumor
or eliminate tumor growth or regrowth than would otherwise result
in the absence of such treatment. Desirably, the amount of
transduced T cells reintroduced into the subject causes an about
10%, about 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about 80%, about 90%, about 95%, about 98%, or about 99%
decrease in tumor size when compared to otherwise same conditions
wherein the transduced T cells are not present.
[0169] Accordingly, the amount of transduced T cells administered
should take into account the route of administration and should be
such that a sufficient number of the transduced T cells will be
introduced so as to achieve the desired therapeutic response.
Furthermore, the amounts of each active agent included in the
compositions described herein (e.g., the amount per each cell to be
contacted or the amount per certain body weight) can vary in
different applications. In general, the concentration of transduced
T cells desirably should be sufficient to provide in the subject
being treated at least from about 1.times.10.sup.6 to about
1.times.10.sup.9 transduced T cells/m.sup.2 (or kg) of a patient,
even more desirably, from about 1.times.10.sup.7 to about
5.times.10.sup.6 transduced T cells/m.sup.2 (or kg) of a patient,
although any suitable amount can be utilized either above, e.g.,
greater than 5.times.10.sup.6 cells/m.sup.2 (or kg) of a patient,
or below, e.g., less than 1.times.10.sup.7 cells/m.sup.2 (or kg) of
a patient. The dosing schedule can be based on well-established
cell-based therapies (see, e.g., U.S. Pat. No. 4,690,915, the
content which is herein incorporated by reference in its entirety),
or an alternate continuous infusion strategy can be employed.
[0170] These values provide general guidance of the range of
transduced T cells to be utilized by the practitioner upon
optimizing the method of the present invention for practice of the
invention. The recitation herein of such ranges by no means
precludes the use of a higher or lower amount of a component, as
might be warranted in a particular application. For example, the
actual dose and schedule can vary depending on whether the
compositions are administered in combination with other
pharmaceutical compositions, or depending on interindividual
differences in pharmacokinetics, drug disposition, and metabolism.
One skilled in the art readily can make any necessary adjustments
in accordance with the exigencies of the particular situation.
[0171] The terms "T cell" or "T lymphocyte" are art-recognized and
are intended to include thymocytes, naive T lymphocytes, immature T
lymphocytes, mature T lymphocytes, resting T lymphocytes, or
activated T lymphocytes. Illustrative populations of T cells
suitable for use in particular embodiments include, but are not
limited to, helper T cells (HTL; CD4+ T cell), a cytotoxic T cell
(CTL; CD8+ T cell), CD4+CD8+ T cell, CD4-CD8- T cell, or any other
subset of T cells. Other illustrative populations of T cells
suitable for use in particular embodiments include, but are not
limited to, T cells expressing one or more of the following
markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127,
CD197, and HLA-DR and if desired, can be further isolated by
positive or negative selection techniques.
[0172] A peripheral blood mononuclear cell (PBMC) is defined as any
blood cell with a round nucleus (i.e., a lymphocyte, a monocyte, or
a macrophage). These blood cells are a critical component in the
immune system to fight infection and adapt to intruders. The
lymphocyte population consists of CD4+ and CD8+ T cells, B cells
and Natural Killer cells, CD14+ monocytes, and
basophils/neutrophils/eosinophils/dendritic cells. These cells are
often separated from whole blood or from leukapheresis products
using FICOLL.TM., a hydrophilic polysaccharide that separates
layers of blood, with monocytes and lymphocytes forming a buffy
coat under a layer of plasma. In one embodiment, "PBMCs" refers to
a population of cells comprising at least T cells, and optionally
NK cells, and antigen presenting cells.
[0173] The term "activation" refers to the state of a T cell that
has been sufficiently stimulated to induce detectable cellular
proliferation. In particular embodiments, activation can also be
associated with induced cytokine production, and detectable
effector functions. The term "activated T cells" refers to, among
other things, T cells that are proliferating. Signals generated
through the TCR alone are insufficient for full activation of the T
cell and one or more secondary or costimulatory signals are also
required. Thus, T cell activation comprises a primary stimulation
signal through the TCR/CD3 complex and one or more secondary
costimulatory signals. Co-stimulation can be evidenced by
proliferation and/or cytokine production by T cells that have
received a primary activation signal, such as stimulation through
the CD3/TCR complex or through CD2.
[0174] As used herein, a resting T cell means a T cell that is not
dividing or producing cytokines. Resting T cells are small
(approximately 6-8 microns) in size compared to activated T cells
(approximately 12-15 microns).
[0175] As used herein, a primed T cell is a resting T cell that has
been previously activated at least once and has been removed from
the activation stimulus for at least about 1 hour, at least about 2
hours, at least about 3 hours, at least about 4 hours, at least
about 5 hours, at least about 6 hours, at least about 12 hours, at
least about 24 hours, at least about 48 hours, at least about 60
hours, at least about 72 hours, at least about 84 hours, at least
about 96 hours, at least about 108 hours, or at least about 120
hours. Alternatively, resting may be carried out within a period of
from about 0.5 hour to about 120 hours, about 0.5 hour to about 108
hours, about 0.5 hour to about 96 hours, about 0.5 hour to about 84
hours, about 0.5 hour to about 72 hours, about 0.5 hour to about 60
hours, about 0.5 hour to about 48 hours, about 0.5 hour to about 36
hours, about 0.5 hour to about 24 hours, about 0.5 hour to about 18
hours, about 0.5 hour to about 12 hours, about 0.5 hour to about 6
hours, about 1 hour to about 6 hours, about 2 hours to about 5
hours, about 3 hours to about 5 hours, or about 4 hours to about 5
hours. Primed T cells usually have a memory phenotype.
[0176] A population of T cells may be induced to proliferate by
activating T cells and stimulating an accessory molecule on the
surface of T cells with a ligand, which binds the accessory
molecule. Activation of a population of T cells may be accomplished
by contacting T cells with a first agent which stimulates a TCR/CD3
complex-associated signal in the T cells. Stimulation of the
TCR/CD3 complex-associated signal in a T cell may be accomplished
either by ligation of the T cell receptor (TCR)/CD3 complex or the
CD2 surface protein, or by directly stimulating receptor-coupled
signalling pathways. Thus, an anti-CD3 antibody, an anti-CD2
antibody, or a protein kinase C activator in conjunction with a
calcium ionophore may be used to activate a population of T
cells.
[0177] To induce proliferation, an activated population of T cells
may be contacted with a second agent, which stimulates an accessory
molecule on the surface of the T cells. For example, a population
of CD4+ T cells can be stimulated to proliferate with an anti-CD28
antibody directed to the CD28 molecule on the surface of the T
cells. Alternatively, CD4+ T cells can be stimulated with a natural
ligand for CD28, such as B7-1 and B7-2. The natural ligand can be
soluble, on a cell membrane, or coupled to a solid phase surface.
Proliferation of a population of CD8+ T cells may be accomplished
by use of a monoclonal antibody ES5.2D8, which binds to CD9, an
accessory molecule having a molecular weight of about 27 kD present
on activated T cells. Alternatively, proliferation of an activated
population of T cells can be induced by stimulation of one or more
intracellular signals, which result from ligation of an accessory
molecule, such as CD28.
[0178] The agent providing the primary activation signal and the
agent providing the costimulatory agent can be added either in
soluble form or coupled to a solid phase surface. In a preferred
embodiment, the two agents may be coupled to the same solid phase
surface.
[0179] Following activation and stimulation of an accessory
molecule on the surface of the T cells, the progress of
proliferation of the T cells in response to continuing exposure to
the ligand or other agent, which acts intracellularly to simulate a
pathway mediated by the accessory molecule, may be monitored. When
the rate of T cell proliferation decreases, T cells may be
reactivated and re-stimulated, such as with additional anti-CD3
antibody and a co-stimulatory ligand, to induce further
proliferation. In one embodiment, the rate of T cell proliferation
may be monitored by examining cell size. Alternatively, T cell
proliferation may be monitored by assaying for expression of cell
surface molecules in response to exposure to the ligand or other
agent, such as B7-1 or B7-2. The monitoring and re-stimulation of T
cells can be repeated for sustained proliferation to produce a
population of T cells increased in number from about 100- to about
100,000-fold over the original T cell population.
[0180] The method of the present disclosure can be used to expand
selected T cell populations for use in treating an infectious
disease or cancer. The resulting T cell population can be
genetically transduced and used for immunotherapy or can be used
for in vitro analysis of infectious agents. Following expansion of
the T cell population to sufficient numbers, the expanded T cells
may be restored to the individual. The method of the present
disclosure may also provide a renewable source of T cells. Thus, T
cells from an individual can be expanded ex vivo, a portion of the
expanded population can be re-administered to the individual and
another portion can be frozen in aliquots for long term
preservation, and subsequent expansion and administration to the
individual. Similarly, a population of tumor-infiltrating
lymphocytes can be obtained from an individual afflicted with
cancer and the T cells stimulated to proliferate to sufficient
numbers and restored to the individual.
[0181] The present disclosure may also pertain to compositions
containing an agent that provides a costimulatory signal to a T
cell for T cell expansion (e.g., an anti-CD28 antibody, B7-1 or
B7-2 ligand), coupled to a solid phase surface which may
additionally include an agent that provides a primary activation
signal to the T cell (e.g., an anti-CD3 antibody) coupled to the
same solid phase surface. These agents may be preferably attached
to beads or flasks or bags. Compositions comprising each agent
coupled to different solid phase surfaces (i.e., an agent that
provides a primary T cell activation signal coupled to a first
solid phase surface and an agent that provides a costimulatory
signal coupled to a second solid phase surface) may also be within
the scope of this disclosure.
[0182] In an aspect, TAA peptides that are capable of use with the
methods and embodiments described herein include, for example,
those TAA peptides described in U.S. Publication 20160187351, U.S.
Publication 20170165335, U.S. Publication 20170035807, U.S.
Publication 20160280759, U.S. Publication 20160287687, U.S.
Publication 20160346371, U.S. Publication 20160368965, U.S.
Publication 20170022251, U.S. Publication 20170002055, U.S.
Publication 20170029486, U.S. Publication 20170037089, U.S.
Publication 20170136108, U.S. Publication 20170101473, U.S.
Publication 20170096461, U.S. Publication 20170165337, U.S.
Publication 20170189505, U.S. Publication 20170173132, U.S.
Publication 20170296640, U.S. Publication 20170253633, U.S.
Publication 20170260249, U.S. Publication 20180051080, and U.S.
Publication No. 20180164315, the contents of each of these
publications and sequence listings described therein are herein
incorporated by reference in their entireties.
[0183] In an aspect, T cells described herein selectively recognize
cells which present a TAA peptide described in one of more of the
patents and publications described above.
[0184] In another aspect, TAA that are capable of use with the
methods and embodiments described herein include at least one
selected from SEQ ID NO: 1 to SEQ ID NO: 157.
TABLE-US-00001 SEQ ID Amino Acid NO: Sequence 1 YLYDSETKNA 2
HLMDQPLSV 3 GLLKKINSV 4 FLVDGSSAL 5 FLFDGSANLV 6 FLYKIIDEL 7
FILDSAETTTL 8 SVDVSPPKV 9 VADKIHSV 10 IVDDLTINL 11 GLLEELVTV 12
TLDGAAVNQV 13 SVLEKEIYSI 14 LLDPKTIFL 15 YTFSGDVQL 16 YLMDDFSSL 17
KVWSDVTPL 18 LLWGHPRVALA 19 KIWEELSVLEV 20 LLIPFTIFM 21 FLIENLLAA
22 LLWGHPRVALA 23 FLLEREQLL 24 SLAETIFIV 25 TLLEGISRA 26 ILQDGQFLV
27 VIFEGEPMYL 28 SLFESLEYL 29 SLLNQPKAV 30 GLAEFQENV 31 KLLAVIHEL
32 TLHDQVHLL 33 TLYNPERTITV 34 KLQEKIQEL 35 SVLEKEIYSI 36
RVIDDSLVVGV 37 VLFGELPAL 38 GLVDIMVHL 39 FLNAIETAL 40 ALLQALMEL 41
ALSSSQAEV 42 SLITGQDLLSV 43 QLIEKNWLL 44 LLDPKTIFL 45 RLHDENILL 46
YTFSGDVQL 47 GLPSATTTV 48 GLLPSAESIKL 49 KTASINQNV 50 SLLQHLIGL 51
YLMDDFSSL 52 LMYPYIYHV 53 KVWSDVTPL 54 LLWGHPRVALA 55 VLDGKVAVV 56
GLLGKVTSV 57 KMISAIPTL 58 GLLETTGLLAT 59 TLNTLDINL 60 VIIKGLEEI 61
YLEDGFAYV 62 KIWEELSVLEV 63 LLIPFTIFM 64 ISLDEVAVSL 65 KISDFGLATV
66 KLIGNIHGNEV 67 ILLSVLHQL 68 LDSEALLTL 69 VLQENSSDYQSNL 70
HLLGEGAFAQV 71 SLVENIHVL 72 YTFSGDVQL 73 SLSEKSPEV 74 AMFPDTIPRV 75
FLIENLLAA 76 FTAEFLEKV 77 ALYGNVQQV 78 LFQSRIAGV 79 ILAEEPIYIRV 80
FLLEREQLL 81 LLLPLELSLA 82 SLAETIFIV 83 AILNVDEKNQV 84 RLFEEVLGV 85
YLDEVAFML 86 KLIDEDEPLFL 87 KLFEKSTGL 88 SLLEVNEASSV 89 GVYDGREHTV
90 GLYPVTLVGV 91 ALLSSVAEA 92 TLLEGISRA 93 SLIEESEEL 94 ALYVQAPTV
95 KLIYKDLVSV 96 ILQDGQFLV 97 SLLDYEVSI 98 LLGDSSFFL 99 VIFEGEPMYL
100 ALSYILPYL 101 FLFVDPELV 102 SEWGSPHAAVP 103 ALSELERVL 104
SLFESLEYL 105 KVLEYVIKV 106 VLLNEILEQV 107 SLLNQPKAV 108 KMSELQTYV
109 ALLEQTGDMSL 110 VIIKGLEEITV 111 KQFEGTVEI 112 KLQEEIPVL 113
GLAEFQENV 114 NVAEIVIHI 115 ALAGIVTNV 116 NLLIDDKGTIKL 117
VLMQDSRLYL 118 KVLEHVVRV 119 LLWGNLPEI 120 SLMEKNQSL 121 KLLAVIHEL
122 ALGDKFLLRV 123 FLMKNSDLYGA
124 KLIDHQGLYL 125 GPGIFPPPPPQP 126 ALNESLVEC 127 GLAALAVHL 128
LLLEAVWHL 129 SIIEYLPTL 130 TLHDQVHLL 131 SLLMWITQC 132 FLLDKPQDLSI
133 YLLDMPLWYL 134 GLLDCPIFL 135 VLIEYNFSI 136 TLYNPERTITV 137
AVPPPPSSV 138 KLQEELNKV 139 KLMDPGSLPPL 140 ALIVSLPYL 141 FLLDGSANV
142 ALDPSGNQLI 143 ILIKHLVKV 144 VLLDTILQL 145 HLIAEIHTA 146
SMNGGVFAV 147 MLAEKLLQA 148 YMLDIFHEV 149 ALWLPTDSATV 150 GLASRILDA
151 SYVKVLHHL 152 VYLPKIPSW 153 NYEDHFPLL 154 VYIAELEKI 155
VHFEDTGKTLLF 156 VLSPFILTL 157 HLLEGSVGV
EXAMPLES
Example 1
[0185] Autologous T Cell Manufacturing Process
[0186] Adoptive cell transfer of purified naive (T.sub.n), stem
cell memory (T.sub.scm), and central memory (T.sub.cm) T cell
subsets causes superior tumor regression compared with transfer of
the more-differentiated effector memory (T.sub.em) and effector
(T.sub.eff) T cells. Traditional manufacturing process for an
engineered T cell product may take 10-15 days long. However, a
process longer than about 12 days, e.g., 14 days, may result in
reduced potency of the cells, e.g., fewer more-effective T.sub.n,
T.sub.scm, and T.sub.cm T cell subsets and more less-effective
T.sub.em and T.sub.eff T cell subsets. For example, FIG. 1A shows
prolonging ex vivo culturing of T cells, e.g., 14 days, from two
healthy donors, e.g., donor 6 and donor 8, in which the desirable
T.sub.cm T cell subsets were reduced from that cultured for 0, 6,
or 10 days. On the other hand, the more differentiated and less
persistent T.sub.em T cell subsets were increased from that
cultured for 0, 6, or 10 days. FIG. 1B shows prolonging ex vivo
culturing of T cells, e.g., 14 days, from three patients, e.g.,
patient 864, patient 453, and patient 265, in which the desirable
T.sub.cm T cell subsets were reduced from that cultured for 0, 6,
or 10 days. On the other hand, the more differentiated and less
persistent T.sub.em T cell subsets were increased from that
cultured for 0, 6, or 10 days.
[0187] Fewer more-effective T.sub.n, T.sub.scm, and T.sub.cm T cell
subsets may result in fewer effectively activated T cells that
secret cytokines, e.g., interferon gamma (INF-.gamma.). FIG. 2
shows reduced INF-.gamma. secretion by peripheral blood mononuclear
cells (PBMC) obtained from three healthy donors, e.g., donor 6
(D6), donor 7 (D7), and donor 8 (D8), activated and cultured for 15
days as compared with that activated and cultured for 10 days.
[0188] To shorten the manufacturing process, embodiments of the
present disclosure include an about 7 to about 10-day process
leading to the manufacturing of over 10 billion (10.times.10.sup.9)
cells without the loss of potency. In addition, the concentrations
of several raw materials may be optimized to reduce the cost of
good by 30%.
[0189] Effect of Eliminating or Modifying Resting Conditions in
Autologous T Cell Manufacturing Process on T Cell Activation
[0190] FIG. 3 shows an experimental design used to test the effect
of resting conditions on T cell activation and expansion. Briefly,
group A represents a first batch of PBMC that were thawed on Day 0,
followed by resting without cytokines overnight (O/N), i.e., 24
hours, followed by activating the rested PBMC with anti-CD3 and
anti-CD28 antibodies immobilized on non-tissue culture treated
plates. IL-7 is a homeostatic cytokine that promotes survival of T
cells by preventing apoptosis. IL-7 may be added to PBMC during
resting. Groups B1-B3 represent a second batch of PBMC that were
thawed on Day 1, followed by resting in the presence of IL-7 (group
B1) or in the presence of IL-7+IL-15 (group B2) or without cytokine
(group B3) for 4-6 hours, followed by activating the rested PBMC
with anti-CD3 and anti-CD28 antibodies immobilized on non-tissue
culture treated plates. Group C represents a third batch of PBMC
that were thawed on Day 1 (without resting and without cytokine),
followed by activating the thawed PBMC with anti-CD3 and anti-CD28
antibodies immobilized on tissue culture plates. Cells may be
harvested and counted on Day 8-10, followed by activation panel
analysis.
[0191] CD25 and CD69 are activation markers on the surface of
cytokine- or mitogen-activated lymphocytes. The binding and entry
of the VSV-G pseudotyped lentiviral vectors, such as LV-R73, has
been shown to be mediated by interaction of the VSV-G envelop
protein with low density lipoprotein receptor (LDL-R) on the host
cells. Resting T cells do not express LDL-R, however activation
with anti-CD3 and anti-CD28 antibodies induces LDL-R expression on
T cells and permits efficient lentiviral transduction. This
suggests that kinetics of LDL-R expression regulated by level of
activation can impact transduction efficiency with VSV-G lentiviral
vector.
[0192] FIG. 4 shows CD25, CD69, and hLDL-R expression levels among
groups A, 131-133, and C are comparable, indicating that the time
for resting may be shortened, e.g., from 24 hours to 4-6 hours,
without significantly reducing T cell activation.
[0193] Effect of Eliminating or Modifying Resting Conditions in
Autologous T Cell Manufacturing Process on T Cell Expansion
[0194] FIGS. 5A and 5B show fold expansion and cell viability in
groups A and B1-B3 are comparable on Day 7 expansion and Day 10
expansion, respectively. Group C, which is without resting,
however, has the least fold expansion on Day 7 expansion (5-fold)
(FIG. 5A) and Day 10 expansion (16-fold) (FIG. 5B). These results
suggest that the time for resting may be shortened, e.g., from 24
hours to 4-6 hours, without significantly reducing T cell
expansion.
[0195] FIGS. 6 and 7 show fold expansion and viability of activated
T cells transduced with a viral vector expressing TCR, e.g.,
LV-R73, in 2 donors, i.e., donor 1 (FIG. 6) or donor 2 (FIG. 7), in
groups A, B1-133, and C on Day 9 expansion. Groups B1 and B2 show
better cell expansion than groups A, B3, and C, indicating that
brief resting time, e.g., 5 hours, in the presence of cytokines,
e.g., IL-7 or IL-7+IL-15, may increase expansion of transduced T
cells. Rep1 and Rep2 represent two replicates. These results
support use of shortened resting time, e.g., from 24 hours to 4-6
hours, in autologous T cell manufacturing process in the presence
of cytokines, e.g., IL-7 and/or IL-15, without significantly
reducing T cell expansion.
[0196] Effect of Eliminating or Modifying Resting Conditions in
Autologous T Cell Manufacturing Process on Transgene Expression in
T Cells
[0197] Using peptide/MHC complex-loaded tetramers to detect T cells
expressing transduced TCR that specifically binds peptide/MHC
complex, FIG. 8 shows comparable transgene expression, e.g.,
recombinant TCR expression, in T cells rested for 4 hours (with
IL-7) and 24h (without cytokine) in a T75 tissue culture flask or 4
hours (with IL-7) in G-Rex 100 flask in a large-scale production
run for donor 13, donor 14, and donor 16. These results suggest
that the resting time may be shortened, e.g., from 24 hours to 4-6
hours in a scale-up manufacturing process, without significantly
reducing transgene expression in transduced T cells. In addition,
use of one G-Rex 100 flask can simplify the resting process further
by replacing multiple T75 flasks.
[0198] FIG. 9 shows comparable fold expansion on Day 10 expansion
in T cells rested for 4-6 hours, 6 hours, or 24 hours in small
scale or large-scale production for donor 13, donor 14, and donor
16. These results suggest that the resting time may be shortened,
e.g., from 24 hours to 4-6 hours in a scale-up manufacturing
process, without significantly impacting the expansion of
transduced T cells.
[0199] Effect of Concentration of Anti-CD3 and Anti-CD28 Antibodies
in Autologous T Cell Manufacturing Process on T Cell Activation
[0200] Activation is an important step in autologous T cell
manufacturing processes because both transduction efficiency and
rate of expansion rely on T cell activation. Stimulation of T cells
via engagement of CD3 receptor and a co-receptor, such as CD28,
using antibodies is a common method of activating T cells. T cell
activation serves as a preparatory step for transduction with viral
vectors, such as lentiviral vector.
[0201] FIG. 10 shows an experimental design used to test the effect
of concentration of anti-CD3 and anti-CD28 antibodies on T cell
activation. Briefly, on Day 0, PBMC were thawed and cultured or
rested without activation cytokines overnight or 24 hours. On Day
1, the rested PBMC were activated by incubating them in two 24-well
plates coated with different concentrations, e.g., 0.1 .mu.g/ml,
0.25 .mu.g/ml, 0.5 .mu.g/ml, 1.0 .mu.g/ml, of anti-CD3 and
anti-CD28 antibodies in the presence of IL-7+IL-15. On Day 2, the
activated T cells were analyzed for CD25, CD69, and hLDL-R
expression and transduced with VSV-G pseudotyped lentiviral
vectors, e.g., 1.times.Eng LV-R73. On Day 6/7 and 9, analyses, such
as cell counts, viability, and fluorescence-activated cell sorting
(FACS) with dextramers (Dex), which are multimers based on a
dextran backbone bearing multiple fluorescein and peptide/MHC
complexes for detecting T cells expressing recombinant TCR, were
performed.
[0202] FIG. 11 shows, prior to viral transduction, T cells
activated with 0.5 .mu.g/ml and 1.0 .mu.g/ml of anti-CD3 and
anti-CD28 antibodies have comparable levels of CD25, CD69, and
hLDL-R expression within each donor 16 and donor 14. However, these
expression levels are significantly higher than those from T cells
activated with lower concentrations, e.g., 0.1 .mu.g/ml and 0.25
.mu.g/ml, of anti-CD3 and anti-CD28 antibodies. These results
suggest that the concentration of anti-CD3 and anti-CD28 antibodies
may be reduced, e.g., from 1.0 .mu.g/ml to 0.5 .mu.g/ml, without
significantly reducing T cell activation.
[0203] Effect of Concentration of Anti-CD3 and Anti-CD28 Antibodies
and Cytokines in Autologous T Cell Manufacturing Process on T Cell
Expansion
[0204] FIG. 12 shows, on Day 10 expansion, cell counts of T cells
activated by 0.5 .mu.g/ml or 1.0 .mu.g/ml of anti-CD3 and anti-CD28
antibodies in the presence of different concentrations, e.g., 25
ng/ml, 50 ng/ml, or 100 ng/ml, of IL-15 are comparable within each
donor 16, donor 13, and donor 14. These results suggest that the
concentration of anti-CD3 and anti-CD28 antibodies may be reduced,
e.g., from 1.0 .mu.g/ml to 0.5 .mu.g/ml, and the concentration of
IL-15 may be reduced, e.g., from 100 ng/ml to 25 ng/ml, without
significantly reducing T cell expansion.
[0205] FIG. 13 shows tetramer staining of recombinant
TCR-transduced T cells activated by 0.5 .mu.g/ml or 1.0 .mu.g/ml of
anti-CD3 and anti-CD28 antibodies in the presence of different
concentrations, e.g., 25 ng/ml, 50 ng/ml, or 100 ng/ml, of IL-15
are comparable within each donor 16, donor 13, and donor 14. These
results suggest that the concentration of anti-CD3 and anti-CD28
antibodies may be reduced, e.g., from 1.0 .mu.g/ml to 0.5 .mu.g/ml,
and the concentration of IL-15 may be reduced, e.g., from 100 ng/ml
to 25 ng/ml, without significantly reducing viral transduction of T
cells.
[0206] Together, these results suggest that (1) resting time after
thawing PBMC can be shortened, e.g., from 24 hours to 4-6 hours,
without significantly reducing T cell activation, transgene
expression, and T cell expansion; and (2) concentrations of
anti-CD3 and anti-CD28 antibodies can be reduced, e.g., from 1.0
.mu.g/ml to 0.5 .mu.g/ml, and concentrations of cytokines can be
reduced, such as IL-15, e.g., from 100 ng/ml to 25 ng/ml, without
significantly reducing T cell activation, transgene expression, and
T cell expansion.
[0207] Effect of duration of activation in autologous T cell
manufacturing process on transduction efficiency with lentiviral
vector
[0208] One of the major goals of developing an autologous T cell
manufacturing process is to improve the rate of transduction
achieved in primary human T cells with the lentiviral construct
encoding TCR. Unlike gamma retroviruses that can transduce only
dividing cells, lentiviruses, in theory, can transduce both
dividing and non-dividing cells. However, transducing resting T
cells with lentiviruses have yielded poor transduction
efficiencies. Activation of T cells has been shown to facilitate
their transduction with a lentivirus. Thus, stimulation of T cells
with anti-CD3 and anti-CD28 antibodies in immobilized, beads or
soluble form, has become a pre-requisite for performing lentiviral
transduction and is a standard part of manufacturing genetically
modified T cells for adoptive cell therapy.
[0209] Because the T cell activation step plays a critical role in
preparing T cells for transduction, the effective duration of
activation with anti-CD3 and anti-CD28 antibodies may need to be
optimized.
[0210] To determine the optimal duration of activation, a time
course study evaluating the effect of different duration of
activation on transduction efficiency was performed. The results
show that optimal window for transducing T cells may be after
16-24h of activation with anti-CD3 and anti-CD28 antibodies. Thus,
time for T-cell activation prior to transduction may be reduced
from 48h to 16-24h for all further process development and clinical
manufacturing.
[0211] In another embodiment of the present disclosure, for fresh
PBMC, i.e., not frozen, resting may not be needed. Thus, fresh
PBMC, without resting, may be activated by anti-CD3 antibody and
anti-CD28 antibody, followed by viral vector transduction to obtain
transduced T cells.
[0212] Although methods of transducing T cells may involve
sequential steps of activating T cells in tissue culture, followed
by transferring the activated T cells to different tissue culture,
in which transducing activated T cells with viral vectors takes
place, activating and transducing steps, however, may be carried
out concurrently. For example, while T cells are being activated by
anti-CD3 and anti-CD28 antibodies, transducing activated T cells
may be carried out simultaneously in the same culture. By doing so,
the entire T cell transducing process, i.e., from providing PBMC to
obtaining transduced T cells, may be shortened to, for example, 3-4
days.
Example 2
[0213] Determine optimal duration of T cell activation for the
improvement of transduction efficiency with lentiviral
construct
[0214] PBMC from healthy donors were activated using anti-CD3 and
anti-CD28 antibodies for different time intervals in preparation
for transduction. Activated T cells from PBMC were treated with
concentrated supernatants generated using different lentiviral
constructs expressing TAA targeting R7P1 D5 TCR. Transduced cells
were expanded in the presence of IL-7 and IL-15. The products were
compared based on R7P1 D5 TCR transgene expression as determined by
flow cytometry using specific dextramer/tetramer.
[0215] Representative Materials and Methods
TABLE-US-00002 Supplies Manufacturer Catalog # TexMACS media
Miltenyi Biotec 130-097-196 Human AB Serum Gemini 100-512 PBS/EDTA
Lonza BE02-017F IL-7 Peprotech 200-07 IL-15 Peprotech 200-15
Anti-CD3 antibody Ebioscience 16-0037-85 Anti-CD28 antibody
Ebioscience 16-0289-85 24-well non-tissue Co-star 3738 culture
plates G-Rex 24-well plate Wilson Wolf 80192M 15 mL Conical Tube
Falcon 352097 50 ml conical tube Corning 430290 5 mL serological
Pipet BD 53300-421 10 mL serological Pipet BD 53300-523 25 ml
serological pipet BD 53300-567 1000 ul pipet tips Rainin 17007954
200 ul pipet tips Rainin 17007961 20 pL pipet tips Rainin 17007957
1.5 mL Microcentrifuge Fisher 02-681-5 Tube AOPI Staining Solution
Nexcelom CS2-0106 PBS without Mg and Ca Lonza 17-516F/24 96 well
plate Corning 3799 P-20 Micropipettor Rainin 17014382 P-200
Micropipettor Rainin 17014391 P-1000 Micropipettor Rainin 17017382
Pipettaid Drummond 193970L T75 flasks BD Falcon BD353136 T25 flask
Corning 430372 Benzonase Sigma E1014 Protamine sulfate McKesson
804514 Lentivirus Lentigen LV-R73, R78, R72, R22 Live/Dead Aqua dye
Thermo Fisher L-34966 ABC Comp Beads Thermo Fisher A10497 CD3-BV421
BD 562426 CD8-APC Biolegend 301014 TAA Tetramer-PE lmmatics N/A
[0216] Representative Methods
[0217] To compare different durations of T cell activation,
representative experiments described herein were carried out
following standard small-scale T cells generation process
involving, for example, 4 steps: thaw/rest, activation,
transduction and expansion.
[0218] Thaw and Rest
[0219] Frozen PBMC from healthy donors (n=3, D3, D4, D9) were
thawed in warm TexMACS media supplemented with 5% human AB serum.
Cells were treated with benzonase nuclease (50U/ml) for 15 minutes
at 37.degree. C., washed, counted, and put to overnight rest in
complete TexMACS media.
[0220] Activation
[0221] On a day when cells are thawed, 24-well non-tissue culture
plates were coated with anti-CD3 and anti-CD28 antibodies diluted
in PBS (1 .mu.g/mL), sealed and incubated overnight at 4.degree. C.
Next day, rested PBMCs were harvested, counted, washed and
resuspended at the concentration of 1.times.10.sup.6/ml. Antibody
solution was aspirated, and wells were washed with complete media
followed by addition of 2.times.10.sup.6 cells to each well.
Activation was carried out at 37.degree. C. for the specified time
intervals.
[0222] Transduction
[0223] Activated T cells were harvested, washed and counted.
Transduction mixtures containing concentrated virus supernatants,
protamine sulfate (10 .mu.g/ml), IL-7 (10 ng/ml) and IL-15 (100
ng/ml) were prepared. For each transduction, 1.0.times.10.sup.6
cells were separated in a sterile microcentrifuge tube and
centrifuged at 400.times.g for 5 minutes. Each cell pellet was
resuspended in 0.5 ml of the transduction mixture corresponding to
a specific MOI. Cell suspension was placed in an appropriately
labelled well of a 24-well G-Rex plate. After 24 hours of
incubation at 37.degree. C. and 5% CO.sub.2, 1.5 ml media
supplemented with IL-7 (10 ng/ml) and IL-15 (100 ng/ml) was added
to each well. Ninety-six-hour post-transduction, transgene
expression was determined by flow cytometry. Multimeric MHC-peptide
complexes (Dextramer or Tetramer) were used to monitor surface
expression of transgenic TCR by FACS.
[0224] Flow Cytometry
[0225] Briefly, 1.0.times.10.sup.6 cells transduced at given
lentiviral Multiplicity of Infection (MOI) were stained following
the work instructions. For tetramer staining, cells were incubated
with 1 .mu.l of TAA tetramer in 50 .mu.l of Flow buffer for 15
minutes at RT in the dark. Tetramer staining was followed by
staining with antibodies for T cells surface markers (e.g., CD3,
CD4, CD8, etc). Samples were acquired with auto-compensation matrix
derived from compensation beads.
[0226] Results
[0227] PBMC obtained from 2 donors (D3 and D4) were activated for
16, 24 and 48 hours using plate-bound anti-CD3 and CD28 antibodies.
Cells were transduced with 3 different lentiviral constructs (R72,
R21, and R22).
[0228] FIG. 14A shows % CD3.sup.+CD8.sup.+Tetramer.sup.+ T cells
gradually decrease with the increase in the duration of activation
in the order of 16h>24h>48h. This order was consistently
observed for both tested constructs and donors.
[0229] A time course study was performed to determine the optimal
duration of activation for viral transduction that may result in
high transgene expression. PBMC from one donor (D9) were activated
with plate-bound anti-CD3 and anti-CD28 antibodies for the
specified duration of activation, e.g., from 0 to 48 hours, and
transduced with each of two different lentiviral constructs
encoding R7P1 D5 TCR, i.e., LV-R73 and LV-R78.
[0230] FIG. 14B shows transgene expression to be highest in cells
activated for 16-20 hours. Results represent level of transgene
expression measured as % CD3.sup.+CD8.sup.+Tetramer.sup.+ T cells
by flow cytometry 96-hour post transduction. The window for optimal
activation was determined to be about 16 to about 20 hours and may
be extended to the maximum for 24 hours to add flexibility to GMP
manufacturing.
[0231] Overall, these results may explain one of the causes of low
transduction rates observed in T cells activated for 48 hours and
show that shorter activation for 16 to 24 hours may be optimal for
performing lentiviral transduction. Although, robust expansion
achieved by 48-hour activation may be impacted by limiting the
activation time to 16 to 24 hours, this change can be considered
highly beneficial for the product and implemented for all further
process development and into the clinical manufacturing.
Example 3
[0232] Like all bioprocesses, scaling up of T cell manufacturing is
a challenging part of the process development. Maintaining T cell
function and quality to preserve product efficacy is of prime
importance through all stages of scale up. For autologous T cell
manufacturing process, the present inventors identified the
critical steps and divided the scale-up into two parts: scale up of
activation may be carried out on a non-tissue culture surface and
scale up of transduction and expansion may be carried out in a
G-Rex device.
[0233] Although beads or soluble antibodies present simpler methods
to activate cells that are easily scalable, autologous T cell
manufacturing processes which use immobilized antibodies on a
non-tissue culture surface (24-well plate) for activation yielded
the best transduction and expansion rates with the lentivirus.
However, harvesting activated cells from multiple 24-well
non-tissue culture antibody coated plates posed to be a laborious,
time-consuming step that added complexity to an otherwise simple
process. Considering activating up to 1.times.10.sup.9 PBMC, for
example, approximately 20 plates and 480 manipulations may be
required to harvest activated cells in each manufacturing run.
[0234] Conventional methods of activating T cells may include an
open-system and a labor-intensive process using either commercially
available beads or non-tissue culture treated 24-well or 6-well
plates coated with anti-CD3 and anti-CD28 antibodies
("plate-bound") at a concentration of 1 ug/mL each. Open system
methods, however, may take a relatively long time, e.g., about 8
hours, to complete. To simplify the open-system and the
labor-intensive process, embodiments of the present disclosure may
include a straightforward process adaptable to a closed-system that
can be combined with containers, e.g., bags, of commercially
available closed system, e.g., G-Rex.TM. system and Xuri.TM. cell
expansion system, resulting in comparable T cell activation
profile, transducibility of T cells, and functionality of the
end-product with that of T cells activated using the conventional
methods. In addition, methods of the present disclosure, e.g.,
flask-bound method, may take a relatively short time, e.g., about 1
hour, to complete, which is about 8 times faster than the
conventional methods.
[0235] Optimizations for developing the autologous T cell
manufacturing process were performed at a small scale using 24-well
non-tissue culture plates for activation and 24-well G-Rex plates
for transduction and expansion. At this scale, 1-2 million T cells
transduced on Day 2 underwent from 30-fold to 40-fold expansion
until Day 10 to yield 30-80 million cells at the time of harvest.
However, the goal of the final process may be to transduce 250-400
million activated cells and expand them to over 10 billion viable
CD3.sup.+ T cells keeping the manufacturing timeline of 10 days.
Therefore, in an aspect, scaling up of entire process is provided
herein.
[0236] For embodiments of the present disclosure including methods
of activating larger number of cells, non-tissue culture treated
T175cm.sup.2 flasks provide a larger surface area and simpler
platform requiring very few manipulations. After optimization, use
of flask-bound antibodies for activating T cells resulted in
expansion and transduction comparable to plate-bound antibodies.
Thus, scale up of the activation step was a major development in
simplifying the autologous T cell manufacturing process for
clinical manufacturing. Based on greater cell numbers, transduction
and expansion were scaled up from G-Rex-24 well plate to G-Rex 100.
Use of G-Rex devices may facilitate nearly linear scale up of
post-activation steps especially in terms of seeding density. Other
parameters, such as number of feeds and splits, may be standardized
to achieve maximum expansion rates and viability. Validation of the
entire scaled up process in full scale PD runs ensure successful
technology transfer of the T Cell Product #1 process in GMP.
[0237] Plate-Bound Versus Flask-Bound
[0238] T cell activation followed by transduction and expansion are
critical steps of T cell manufacturing. To optimize the conditions
for scaling up of these steps, use of T175cm.sup.2 flasks presented
a suitable platform for activation with larger surface area and
fewer manipulations to replace 24-well plates coated with anti-CD3
and anti-CD28 antibodies. In a comparative study following
optimization of critical parameters in T175cm.sup.2 flasks, cells
activated using antibodies coated on a flask (flask-bound) showed
comparable levels of activation, transduction, and expansion to
cells activated using antibodies coated on 24-well plates
(plate-bound) in multiple donors.
[0239] Further, transduction and expansion steps were scaled up
from small scale (G-Rex-24 well plate) to mid-scale (2-6 G-Rex10 or
1 G-Rex100) to full scale (5-8 G-Rex100). The entire scaled-up
process may be validated in 2 full scale Process Development (PD)
runs. All products generated using the final process passed the
clinical release criteria in terms of % Dex.sup.+
CD3.sup.+CD8.sup.+ cells and generated cell numbers sufficient to
meet the clinical doses.
[0240] Comparison Between T Cells Activated by the Plate-Bound
Method and the Flask-Bound Method (a Non-Tissue Culture Treated
Flask is Coated with Anti-CD3 and Anti-CD28 Antibodies) with
Respect to Activation Level (Flow Cytometry), Transducibility
(Dextramer Staining, FACS), Expansion (Cell Counts), and
Functionality (IFN-.gamma. ELISA)
[0241] PBMC from healthy donors were activated using anti-CD3 and
anti-CD28 antibodies using non-tissue culture treated T175cm.sup.2
flasks or 24-well plates in preparation for transduction. Activated
T cells were transduced with a lentiviral construct encoding R7P1
D5 TCR and seeded in G-Rex 24-well plates or G-Rex10/G-Rex100
flasks. Transduced T cells were expanded in the presence of IL-7
and IL-15 and harvested on Day 10 of the process. In-process and
final testing were performed on the products to determine cell
counts, viability and percentage of transduced CD8.sup.+ T
cells.
[0242] Representative Materials and Methods
TABLE-US-00003 Supplies Manufacturer Catalog # TexMACS media
Miltenyi Biotec 130-097-196 Human AB Serum Gemini 100-512 IL-7
Peprotech 200-07 IL-15 Peprotech 200-15 Anti-CD3 antibody
Ebioscience 16-0037-85 Anti-CD28 antibody Ebioscience 16-0289-85
24-well non-tissue culture plates Co-star 3738 T175 cm.sup.2
non-tissue culture plates Corning 431466 G-Rex 24-well plate Wilson
Wolf 80192M G-Rex10 Wilson Wolf 80040S G-Rex100 Wilson Wolf 80500S
15 mL Conical Tube Falcon 352097 50 ml conical tube Corning 430290
5 mL serological Pipet BD 53300-421 10 mL serological Pipet BD
53300-523 25 ml serological pipet BD 53300-567 1000 ul pipet tips
Rainin 17007954 200 ul pipet tips Rainin 17007961 20 .mu.L pipet
tips Rainin 17007957 1.5 mL Microcentrifuge Tube Fisher 02-681-5
AOPI Staining Solution Nexcelom CS2-0106 PBS without Mg and Ca
Lonza 17-516F/24 96 well plate Corning 3799 P-20 Micropipettor
Rainin 17014382 P-200 Micropipettor Rainin 17014391 P-1000
Micropipettor Rainin 17017382 Pipettaid Drummond 193970L T75 flasks
BD Falcon BD353136 Benzonase Sigma E1014 Protamine sulfate McKesson
804514 Lentivirus Lentigen LV-R73, R78 Live/Dead Aqua dye Thermo
Fisher L-34966 ABC Comp Beads Thermo Fisher A10497 CD3-BV421 BD
562426 CD8-APC Biolegend 301014 CD4-PerCPCy5.5 BD 560650 TAA
Dextramer-PE Immudex N/A
[0243] Methods
[0244] Experiments were carried out following the standard
autologous T cell manufacturing process involving 4 steps:
thaw/rest, activation, transduction, and expansion, however at
different scales.
[0245] Thaw and Rest
[0246] Frozen PBMC from healthy donors were thawed in warm TexMACS
media supplemented with 5% human AB serum (complete media). Cells
were treated with benzonase nuclease (50U/ml) for 15 minutes at
37.degree. C., washed, counted and put to overnight rest in
complete TexMACS media.
[0247] Activation
[0248] On the day of thawing cells, 24-well non-tissue culture
plates or T175cm.sup.2 flasks were coated with anti-CD3 and
anti-CD28 antibodies diluted in PBS (1 .mu.g/mL), sealed and
incubated overnight at 4.degree. C. Next day, rested PBMCs were
harvested, counted, washed and resuspended at the concentration of
1.times.10.sup.6/ml. Antibody solution was aspirated, and wells
were washed with complete media followed by addition of
2.times.10.sup.6 cells to each well. Activation was carried out at
37.degree. C. for the specified time intervals.
[0249] Transduction
[0250] Activated T cells were harvested, washed and counted.
Transduction mixtures containing preclinical lentiviral
supernatants (calculated based on a specified MOI), protamine
sulfate (10 .mu.g/ml) and IL-7 (10 ng/mL) and IL-15 (100 ng/mL).
For each transduction, activated cells were separated and
centrifuged at 400.times.g for 5 minutes. Each cell pellet was
resuspended in the transduction mixture (1 ml per 2.times.10.sup.6
cells) and seeded in an appropriately sized G-Rex flask. After 24
hours of incubation at 37.degree. C. and 5% CO.sub.2, culture
volume in each G-Rex flask was brought to half or full capacity as
specified using media supplemented with IL-7 (10 ng/ml) and IL-15
(100 ng/ml). Cell counts and viability were monitored regularly up
to Day 10 of the process. Multimeric MHC-peptide complexes
(Dextramer or Tetramer) were used to monitor surface expression of
transgenic TCR by FACS.
[0251] Flow Cytometry
[0252] Briefly, 1.0.times.10.sup.6 transduced cells were stained
following the work instructions. Tetramer staining was followed by
staining with antibodies for T cells surface markers. Samples were
acquired with auto-compensation matrix derived from compensation
beads.
[0253] Results
[0254] To evaluate non-tissue culture treated T175cm.sup.2 flasks
as an alternative to 24-well plates for coating anti-CD3 and
anti-CD28 antibodies to activate T cells, the antibody
concentration was kept the same as in small scale, other parameters
such as coating volume, cell density, and seeding volume were
optimized for a larger area in a flask.
[0255] To compare viability and expression of activation markers
CD25 and CD69 and LDL-R in plate-bound (PB) and flask-bound (FB)
activated PBMC, FACS staining and acquisition were performed 16-24
hours post-activation using FB or PB anti-CD3 and CD28 antibodies.
Unstimulated PBMC were used as negative controls.
[0256] FIG. 15 shows, under optimized conditions, T cells activated
in T175cm.sup.2 flasks (flask-bound, FB) exhibit comparable
expression levels of activation markers CD25 and CD69 and LDL-R to
that of T cells activated under plate-bound (PB) conditions. These
results suggest scale-up activation using FB antibodies may be
feasible in view of the comparable levels of activation resulting
from FB and PB activated T cells.
[0257] To compare transgene expression and expansion in Day 10
harvested T cell products (from donor 6, donor 7, and donor 8)
using PB or FB antibodies for activation, surface expression of
R7P1 D5 TCR was determined by flow cytometry using TAA specific
dextramer or transgenic TCR 13 chain specific antibody. Fold
expansion was calculated on the basis of viable cell number seeded
in the G-Rex plate or flask at the time of transduction (Day 2) and
the day of harvest (Day 10). FIGS. 16A and 16B show comparable
levels of transduction and fold expansion, respectively, in FB and
PB activated T cells. These results suggest scale-up transduction
and expansion using FB antibodies may be feasible in view of the
comparable levels of transduction and expansion resulting from FB
and PB activated T cells.
[0258] For further validation of successful scale up of activation,
functionality of T cell products generated by FB and PB activation
methods were compared. To evaluate induction of antigen specific
IFN-.gamma. by LV-R73 transduced T cell products generated using PB
or FB antibodies for activation, IFN-.gamma. released in the
supernatant of T cell co-cultured with tumor cell lines (Target+ve,
Target-ve) was quantitated using a commercially available ELISA
kit.
[0259] FIG. 17 shows FB activated LV-R73 transduced T cells
secreted comparable levels of antigen specific IFN-.gamma. to that
of PB activated transduced T cells in response to tumor cells
expressing TAA in each donor 6, donor, 7, and donor 8. These
results suggest scale-up of IFN-.gamma.-secreting T cells using FB
antibodies may be feasible in view of the comparable levels of
IFN-.gamma. secretion resulting from FB and PB activated T
cells.
[0260] For scale up of the remaining process,
2.5.times.10.sup.8-4.0.times.10.sup.8 activated T cells were
transduced and seeded at optimal seeding density of
0.5.times.10.sup.6 per cm.sup.2 of surface area of the G-Rex100
flask. Multiple G-Rex100 flasks were used to seed the transduced
cells at the optimal density. Additional parameters, such as
conditions for feeding and splitting the cells, were also optimized
to achieve maximum expansion. The final manufacturing process was
tested in 2 full scale Process Development (PD) runs. All products
generated using the final process passed the % Dextramer and
integration copy number release criteria. Cell numbers generated in
these manufacturing runs met clinical dose at all cohort levels.
Results of PD scale up runs are summarized in the Table 1
below.
TABLE-US-00004 TABLE 1 Summary of product characterization from 2
full scale PD runs performed Scale up % % % Integration Run# Donor
CD3 CD8 Dextramer.sup.+ Copy# Cell # 1 Donor 6 99.5% 66.9% 21.6%
0.96 1.01 .times. 10.sup.10 2 Donor 9 96.2% 57.7% 23.1% 1.21 1.05
.times. 10.sup.10
[0261] GMP manufacturing with the above process have yielded over
20 billion cells for a few donors.
[0262] Flask-Bound Versus Bag-Bound
[0263] Comparison Between T Cells Activated by Flask-Bound Method
and Bag-Bound Method (e.g., Saint-Gobain VueLife AC Bag Coated with
Anti-CD3 and Anti-CD28 Antibodies) with Respect to Activation Level
(Flowcytometry), Transducibility (Dextramer Staining, FACS), and
Expansion (Cell Counts)
[0264] To compare activation of T cells using anti-CD3 and
anti-CD28 antibody coated bags versus plates, FIG. 18 shows the
experimental design used to test the effect of anti-CD3 and
anti-CD28 antibody coated bags and plates on T cell activation.
Briefly, on Day 0, PBMC were thawed and rested overnight (24
hours). On Day 1, the rested PBMC were activated by seeding them on
flasks, e.g., T175cm.sup.2 flasks, or bags, e.g., Saint-Gobain
VueLife AC Bags, coated with anti-CD3 and anti-CD28 antibodies for
16-20 hours. On Day 2, activated T cells were analyzed for CD25,
CD69, and hLDL-R expression and transduced with VSV-G pseudotyped
lentiviral vectors, e.g., 1.times.Eng LV-R73. On Day 6/7 and 10,
analyses, such as cell counts, viability, and
fluorescence-activated cell sorting (FACS) with dextramers (Dex),
were performed.
[0265] FIG. 19 shows, under optimized conditions, T cells (from
donor 16 and donor 14) activated in bags bound with antibodies at
concentrations of 1 .mu.g/ml or 2 .mu.g/ml exhibit comparable
expression of activation markers CD25 and CD69 and hLDL-R
expression to those activated under flask-bound (T175cm.sup.2
flask, labelled as "standard") conditions. These results suggest
scale-up activation using bag-bound (Bag) antibodies may be
feasible in view of the comparable levels of activation resulted
from bag-bound (Bag) and flask-bound (FB) activated T cells.
[0266] FIGS. 20 and 21 shows, on Day 6 and Day 10 of expansion,
respectively, T cells (from donor 16 and donor 14) activated in
bags bound with antibodies at concentrations of 1 .mu.g/ml or 2
.mu.g/ml exhibit comparable T cell counts, i.e., cell expansion,
with that of T cells activated under FB conditions. These results
suggest scale-up expansion using bag-bound antibodies may be
feasible in view of the comparable levels of expansion resulting
from bag-bound and flask-bound activated T cells.
Example 4
[0267] Short Rest Versus Overnight Rest in T Cell Manufacturing
Process
[0268] FIG. 22 shows a T cell manufacturing process 220 by resting
PBMC for a period of time of about 4 hours according to one
embodiment of the present disclosure. For example, a T cell
manufacturing process 220 may include Isolation and
cryopreservation of PBMC from leukapheresis (221), in which
sterility may be tested; thaw, rest (e.g., about 4 hours) and
activate T cells (222); transduction with a viral vector (223);
expansion with cytokines (224); split/feed cells (225), in which
cell count and immunophenotyping may be tested; harvest and
cryopreservation of drug product cells (226), in which cell count
and mycoplasma may be tested, and post-cryopreservation release
(227), in which viability, sterility, endotoxin, immunophenotyping,
copy number of integrated vector, and vesicular stomatitis virus
glycoprotein G (VSV-g) may be tested.
[0269] Table 2 shows characteristics of T cells manufactured by
three different qualification runs of T cell manufacturing process
220 by resting PBMC for a short period of time, e.g., about 4 to
about 6 hours, in the presence of IL-7 according to one embodiment
of the present disclosure.
TABLE-US-00005 TABLE 2 Qualification runs (QR) of T cell
manufacturing process 220 by resting PBMC for a short period of
time, e.g., about 4 to about 6 hours, preferably about 4 hours (in
GMP cleanroom) QR1 QR2 QR3 Average % CD3+ 99.6 99.7 99.8 99.7 %
CD8+ 33.5 51.5 75.2 53.4 % Dex+/CD3+CD8+ 35.5 72.7 83.0 63.7 %
Viability 92.0 92.2 91.7 92.0 Residual VSV-g <50 <50 <50
<50 copies/.mu.g copies/.mu.g copies/.mu.g copies/.mu.g Average
copy 1.0 3.0 4.2 2.7 number (per cell) Total viable cells 24.8
.times. 10.sup.9 32.2 .times. 10.sup.9 26.8 .times. 10.sup.9 28.0
.times. 10.sup.9 Transduced cells 2.95 .times. 10.sup.9 12.1
.times. 10.sup.9 16.73 .times. 10.sup.9 10.6 .times. 10.sup.9 Days
manufacturing 10 8 8 8.7 Cells at transduction 281 .times. 10.sup.6
400 .times. 10.sup.6 400 .times. 10.sup.6 360 .times. 10.sup.6
(max) (max) Fold expansion 88-fold 81-fold 67-fold 78.7-fold LV
batch ENG ENG GMP NA
[0270] FIG. 23A and Table 2 show average fold expansion of T cells
(n=7) manufactured by resting PBMC overnight is about
78.7-fold.
[0271] To determine whether transduced TCR is expressed on the cell
surface of expanded T cells, expanded T cells were stained with
peptide/MHC complex-loaded dextramers that specifically bind to
transduced TCR, followed by flow cytometry to identify CD8+ T cells
expressing transduced TCR. FIG. 23B and Table 2 show average %
Dex+/CD8.sup.+ T cells (n=7) manufactured by short rest is about
53.4%, indicating that transduced TCR is expressed on the cell
surface of expanded T cells.
[0272] To determine what T cell phenotypes are present in expanded
T cells expressing transduced TCR, cells were stained with various
immune cell surface markers, followed by flow cytometry to identify
T cell phenotypes, e.g., T.sub.n/scm, T.sub.cm, T.sub.em, and
T.sub.eff. Among them, T.sub.n/scm may be more desirable for
immunotherapy than others because T.sub.n/scm may have properties
of lymphoid homing, proliferation potential, self-renewal, and
multipotency. FIG. 23C shows average about 50% of expanded T cells
expressing transduced TCR (n=4) exhibiting T.sub.n/scm
phenotypes.
[0273] To determine cytotoxic activity of expanded T cells
expressing transduced TCR, tumor cells pulsed with different
concentration of target peptide were incubated with expanded T
cells expressing transduced TCR that specifically recognizes target
peptide/MHC complex, followed by measuring tumor cell growth. FIG.
23D shows expanded T cells expressing transduced TCR inhibit tumor
cell growth in a peptide concentration dependent manner.
[0274] Cytotoxic activities of expanded T cells expressing
transduced TCR appear comparable between PBMC obtained from
different healthy donors, e.g., Donors 7, 13, 17, 18, and 21 (FIG.
23E), and that obtained from different patients, e.g., Patients
312, 319, 351, 472, and 956 (FIG. 23F).
[0275] To determine cytotoxic potential of the expanded T cells
expressing transduced TCR, tumor cells expressing target peptide
were incubated with expanded T cells expressing transduced TCR
(220-T) that specifically recognize target peptide/MHC complex,
followed by measuring fold growth of tumor cells. FIG. 23G and FIG.
23H show increased regression or suppression of tumor growth by
incubation with expanded T cells expressing transduced TCR (220-T)
(effectors) at effectors to tumor cells ratios of 10:1 and 3:1 as
compared with that of the non-transduced T cells lacking
target-specific TCR (NT).
[0276] FIG. 24 shows a T cell manufacturing process 240 by resting
PBMC overnight (about 16 hours). For example, T cell manufacturing
process 240 may include isolation of PBMC (241), in which PBMC may
be used fresh or stored frozen till ready for use, or may be used
as starting materials for T cell manufacturing and selection of
lymphocyte populations (e.g., CD8, CD4, or both) may also be
possible; thaw and rest lymphocytes overnight, e.g., about 16
hours, (242), which may allow apoptotic cells to die off and
restore T cell functionality (this step may not be necessary, if
fresh materials are used); activation of lymphocytes (243), which
may use anti-CD3 and anti-CD28 antibodies (soluble or surface
bound, e.g., magnetic or biodegradable beads); transduction with
CAR or TCR (244), which may use lentiviral or retroviral constructs
encoding CAR or TCR or may use non-viral methods; and expansion of
lymphocytes, harvest, and cryopreservation (245), which may be
carried out in the presence of cytokine(s), serum (ABS or FBS),
and/or cryopreservation media.
[0277] Table 3 shows characteristics of T cells manufactured by
three different qualification runs of a T cell manufacturing
process (240) by resting PBMC overnight, e.g., about 16 hours.
TABLE-US-00006 TABLE 3 Qualification runs (QR) of a T cell
manufacturing process by resting PBMC overnight (in GMP cleanroom)
QR1 QR2 QR3 Average % CD3+ 99.2 99.6 99.7 99.5 % CD8+ 47.9 46.9
60.9 51.9 % Dex+/CD3+CD8+ 36.7 57.3 64.9 53.0 % Viability 85.6 86.8
85.5 86.0 Residual VSV-g <50 <50 <50 <50 copies/.mu.g
copies/.mu.g copies/.mu.g copies/.mu.g Average copy 2.7 3.2 3.6 3.2
number (per cell) Total viable cells 8.7 .times. 10.sup.9 24.3
.times. 10.sup.9 14.2 .times. 10.sup.9 15.7 .times. 10.sup.9
Transduced cells 1.3 .times. 10.sup.9 5.65 .times. 10.sup.9 4.8
.times. 10.sup.9 3.9 .times. 10.sup.9 Days manufacturing 8 9 8 8.3
Cells at transduction 231 .times. 10.sup.6 400 .times. 10.sup.6 400
.times. 10.sup.6 344 .times. 10.sup.6 Fold expansion 38-fold
61-fold 36-fold 45-fold LV batch ENG ENG GMP NA
[0278] In contrast to T cell manufacturing process with short rest,
e.g., about 4 hours, T cells manufactured with rest of about 16
hours yielded less fold expansion of T cells. FIG. 25A and Table 3
show average fold expansion of T cells (n=7) manufactured by
resting PBMC for about 16 hours is about 45-fold, as compared with
about 78.7-fold with short rest of about 4 hours (Table 2). TT and
PQ stand for Technology Transfer and Process Qualification runs,
respectively.
[0279] Overnight rest (about 16 hours) yielded less expanded T
cells expressing transduced TCR than rest of about 4 hours. FIG.
25B and Table 3 show average % Dex+/CD8+ T cells (n=7) manufactured
by resting PBMC overnight for about 16 hours is about 51.9%, as
compared with about 53.4% with rest of about 4 hours (Table 2).
[0280] Overnight rest of about 16 hours yielded less expanded T
cells expressing transduced TCR with T.sub.n/scm phenotype than
rest of about 4 hours. FIG. 25C shows average about 40% of expanded
T cells (n=5) having T.sub.n/scm phenotypes, as compared with about
50% with rest of about 4 hours (FIG. 23C).
[0281] FIG. 25D shows significantly more inhibition of tumor cell
growth by incubation of tumor cells with expanded T cells
expressing transduced TCR (effectors) at effectors to tumor cells
ratios of 10:1, 3:1, and 1:1 than that of the negative controls,
e.g., tumor cells incubated with either expanded T cells that do
not express transduced TCR (Target-ve) or no effectors. In
addition, cytotoxic activities of expanded T cells expressing
transduced TCR appear comparable between PBMC obtained from healthy
donors (n=5) (FIG. 25E) and that obtained from cancer patients
(n=7) (FIG. 25F).
[0282] Table 4 summarizes characteristics of T cells manufactured
with short rest of about 4 hours according to one embodiment of the
present disclosure (process 220) and that with overnight rest of
about 16 hours (process 240).
TABLE-US-00007 TABLE 4 % % Dex+ % Live CD8+ of Fold Harvest
Viability .gtoreq. CD3+ .gtoreq. of CD8+ .gtoreq. Process Expansion
Count 70% 80% CD3+ 10% 220 78.7 28.0 .times. 10.sup.9 92.0 99.7
53.4 63.7 240 45.0 15.7 .times. 10.sup.9 86.0 99.5 51.9 53.0
[0283] Table 4 shows process with short rest 220 (about 4-6 hours)
may allow an extra day in expansion, e.g., Day 8 of process 240 is
Day 9 for process 220, thus, resulting in more cells.
Example 5
[0284] T Cell Manufacturing in Closed System
[0285] As noted above, processes 220 and 240 may be carried out in
open systems, such as G-Rex.TM.. Ex vivo manipulation of
haematopoietic cells, e.g., T cells, in open systems, however, may
introduce risk of contamination with infectious agents and may
reduce engraftment potential and haematopoietic fitness. In
manufacturing clinical cell products, closed cell culture systems
may be preferred due to the assurance of sterility throughout
culture processes.
[0286] FIG. 26 shows ex vivo manipulation protocol in open and
closed systems. Closed systems not only can mitigate external
processing risks and contamination, but also promote product
robustness and quality, and increase product security, thus, can
reduce challenges for downstream processing, final product
analysis, and testing. While relatively small numbers of cells,
e.g., .ltoreq.1.times.10.sup.9, may be cultured in a relatively
small volume in open system, e.g., 1 liter, relatively large
numbers of cells, e.g., from about 1.times.10.sup.9 to about
2.times.10.sup.11, may be cultured in a relatively large volume in
closed system, e.g., from 5 liters (e.g., WAVE (XURI.TM.)
Bioreactor bag and G-Rex.TM. flask) to 50 liters (e.g., static
bag). These closed system cell culturing technologies may deliver
high quality, individualized cell therapies as a regulated, faster,
and cost-effective route of cell manufacturing.
[0287] T cell manufacturing process of the present disclosure may
be carried out in any cell culture closed systems including
commercially available systems, e.g., CliniMACS Prodigy.TM.
(Miltenyi), WAVE (XURI.TM.) Bioreactor (GE Biosciences) alone or in
combination with BioSafe Sepax.TM. II, and G-Rex/GatheRex.TM.
closed system (Wilson Wolf) alone or in combination with BioSafe
Sepax.TM. II. G-Rex.TM.-closed system is the expansion vessel and
GatheRex.TM. is the pump for concentrating and harvesting.
[0288] CliniMACS Prodigy.TM. (Miltenyi)
[0289] CliniMACS Prodigy.TM. with TCT process software and the
TS520 tubing set may allow closed-system processing for cell
enrichment, transduction, washing and expansion. For example,
MACS-CD4 and CD8-MicroBeads may be used for enrichment, TransACT
beads, e.g., CD3/CD28 reagents, may be used for activation,
lentiviral vectors expressing a recombinant TCR may be used for
transduction, TexMACS medium-3%-HS-IL2 for culture and
phosphate-buffered saline/ethylenediaminetetraacetic acid buffer
for washing. This system may yield about 4-5.times.10.sup.9 cells,
contain automated protocols for manufacturing with chamber maximum
.about.300 mL fill volume, and perform selection and activation
(TransACT beads), transduction, and expansion over a 10 to 14-day
process.
[0290] WAVE (Xuri.TM.) Bioreactor (GE Biosciences)
[0291] WAVE (Xuri.TM.) Bioreactor allows T cells to be cultured in
culture bags, e.g., Xuri Cellbags, with and/or without perfusion.
Medium bag for feeding may be 5-liter Hyclone Labtainer. Waste bag
may be Mbag (purchased from GE Healthcare). This system may yield
about 15-30.times.10.sup.9 cells, use unicorn software that allows
for culture control and monitoring, contain rocking tray that may
hold from about 0.3-liter to about 25 liters, and perform perfusion
function to maintain culture volume while mediating gas exchange
and introducing fresh media and cytokines to cell culture.
[0292] WAVE (Xuri.TM.) Bioreactor may include Xuri Bags for
expansion, Saint Gobain's VueLife bags for thawing and resting, and
VueLife AC bags for activation. WAVE (Xuri.TM.) Bioreactor may be
used in combination with other technologies, e.g., Sepax.TM. cell
separation system (GE Biosciences) for culture washing and volume
reduction steps. Sterile welder (Terumo BCT.TM.) may be used for
connecting sterile bags for solution transfer and heat sealer for
sealing tubing.
[0293] Sepax.TM. cell separation system relies on a separation
chamber that provides both separation through rotation of the
syringe chamber (centrifugation) and component transfer through
displacement of the syringe piston. An optical sensor measures the
light absorbency of the separated components and manages the flow
direction of each of them in the correct output container, for
example, plasma, buffy coat, and red blood cells may be thus
separated and collected from blood samples.
[0294] FIG. 27 shows, on Day 0, frozen PBMC isolated by Sepax.TM.
cell separation system may be thawed, washed, rested, e.g.,
overnight (O/N), and culture bags, e.g., VueLife AC cell bags, may
be coated with anti-CD3 antibody and anti-CD28 antibody; on Day 1,
rested PBMC may be transferred to culture bags coated with anti-CD3
antibody and anti-CD28 antibody for activation; on Day 2, cells may
be washed and media may be reduced by Sepax.TM. cell separation
system to an appropriate volume suitable for viral transduction,
e.g., transduced with lentiviral vector expressing TCR. Cell
expansion can be performed in Xuri.TM. culture bags on a rocking
tray with perfusion function to maintain culture volume while
mediating gas exchange and introducing fresh media and cytokines to
cell culture. Expanded transduced T cells may be harvested and
washed using Sepax.TM. cell separation system.
[0295] G-Rex/GatheRex.TM. Closed System (Wilson Wolf)
[0296] G-Rex/GatheRex.TM. closed system comprises a gas-exchange
vessel (G-Rex-CS) for cell expansion and an automated pump
(GatheRex) that may allow the operator to drain the excess media
present in the culture and collect cells without risk of
contamination. The harvesting process may be divided into two
stages: cell concentrating and cell harvesting. In cell
concentrating process, GatheRex.TM. closed system may operate via
an air pump, which pressurizes the G-Rex.TM. device, e.g., flasks,
with sterile air, allowing 90% of the medium residing above the
cells to be displaced into a medium collection bag. Once this
process is complete, a first optical detector senses the presence
of air in the medium collection line, automatically stopping the
pump. Prior to beginning the harvest process, the operator may
resuspend the cells using the residual 10% of the medium by
manually swirling the G-Rex.TM. device to dislodge cells from the
gas-permeable membrane. The air pump is then reactivated, and the
resuspended cells are drawn into the cell collection bag. This
phase may automatically end once a second optical detector detects
air in the cell collection line. This system may yield about
15-20.times.10.sup.9 cells and hold 5-liters per vessel.
[0297] G-Rex/GatheRex.TM. closed system may support transduction
and expansion in the vessel and harvest with the pump. Thawing,
resting, and activation steps may be carried out in VueLife.TM.
bags. GatheRex.TM. closed system may be used in combination with
other technologies, e.g., Sepax.TM. cell separation system for
culture washing and volume reduction stepsSterile welder (Terumo
BCT.TM.) may be used for connecting sterile bag for solution
transfer and heat sealer for sealing tubing.
[0298] FIG. 28 shows on Day 0, frozen PBMC isolated by Sepax.TM.
cell separation system may be thawed, washed, rested, e.g.,
overnight (O/N); on Day 1, culture bags may be coated with anti-CD3
antibody and anti-CD28 antibody and rested PBMC may be transferred
to the coated culture bags for activation; on Day 2, cells may be
washed and media may be reduced by Sepax.TM. cell separation system
to an appropriate volume suitable for viral transduction, e.g.,
transduced with lentiviral vector expressing TCR. Cell expansion
and feeding may be performed in G-Rex.TM. closed system devices.
Expanded transduced T cells may then be harvested using the
GatheRex.TM. pump and washed using Sepax.TM. cell separation
system.
[0299] Table 5 shows comparison between T cells obtained by open
systems, e.g., G-Rex.TM., as shown in Table 4, i.e., processes 220
and 240, and T cells obtained by closed systems, e.g., CliniMACS
Prodigy.TM., WAVE (XURI.TM.) Bioreactor in combination with BioSafe
Sepax.TM. II, and G-Rex/GatheRex.TM. closed system in combination
with BioSafe Sepax.TM. II.
TABLE-US-00008 TABLE 5 % Live % Dex+ of Fold Viability .gtoreq.
CD3+ .gtoreq. % CD8+ CD8+ .gtoreq. Process Expansion Harvest Count
70% 80% of CD3+ 10% 220 78.7 28.0 .times. 10.sup.9 92.0 99.7 53.4
63.7 240 45.0 15.7 .times. 10.sup.9 86.0 99.5 51.9 53.0 CliniMACS
55.0 4.4 .times. 10.sup.9 95.4 98.5 55.0 39.7 Prodigy .TM. WAVE
40.3 16.1 .times. 10.sup.9 92.0 99.6 60.8 41.7 (XURI .TM.)
Bioreactor in combination with BioSafe Sepax .TM. II G-Rex/ 46.3
18.5 .times. 10.sup.9 89.7 99.4 62.8 49.5 GatheRex .TM. in
combination with BioSafe Sepax .TM. II
[0300] These results show T cell manufacturing process of the
present disclosure can be readily performed in closed systems to
produce T cells with comparable characteristics to that produced in
open systems, while mitigating external processing risks and
contamination, promoting product robustness and quality, and
increasing product security, and thus, reducing challenges for
downstream processing, final product analysis, and testing.
[0301] To further compare functional characteristics of engineered
T cells manufactured in closed systems with that manufactured in
open systems, PBMCs obtained from donor 17 were processed to
produce expanded transduced T cells according to the process of the
present disclosure. The expanded transduced T cells expressing TCR
were then measured for IFN-.gamma. release in the present or
absence of TCR-specific peptide/MHC complex (target).
[0302] FIG. 29 shows that engineered T cells manufactured in closed
systems as measured by two runs, Run #1 and Run #2, released
significantly more IFN-.gamma. in the presence of target than that
manufactured in open system, e.g., process 220. These results
suggest that engineered T cells manufactured in closed systems may
exhibit greater cytotoxic activity than that manufactured in open
systems.
Example 6
[0303] GMP Manufacturing of TCR-Engineered T Cells in about 5 to 6
Days
[0304] Adoptive cellular therapy with autologous engineered T cells
approach capitalizes on translational development of safe and
effective targets and their cognate TCRs. These TCRs are
genetically engineered into patients' own (autologous) T-cells for
the immunotherapy of solid tumors.
[0305] FIG. 30 shows manufacturing outline of three T-cell products
(T Cell Product #1, T Cell Product #2, and T Cell Product #3) each
expressing a transgenic TCR against its own respective HLA-A*02:01
restricted tumor targeted antigen. T Cell Product #1 and T Cell
Product #2 were manufactured in about 8-11 days and about 7-10
days, respectively, from thawing frozen PBMC, resting the thawed
PBMC, and activating the rested PBMC (Step 2), transducing the
activated T cells (Step 3), to "harvest and cryopreservation of
drug product cells" (Step 6), using open systems for IND driven
phase 1 first in man trials.
[0306] T Cell Product #3 may be manufactured by shortening the
expansion phase from about 5-8 days (T Cell Products #1 and #2) to
about 3-4 days. In addition, T Cell Product #3 may be manufactured
by activating fresh PBMC, i.e., PBMC is not cryopreserved and then
thawed, on Day 0. This is in contrast to the manufacturing T Cell
Products #1 by thawing the cryopreserved PBMC on Day 0 and then
activating the thawed PBMC on Day 1 and the manufacturing T Cell
Products #2 by thawing the cryopreserved PBMC and activating the
thawed PBMC on Day 0.
[0307] In contrast to T Cell Products #1 and #2, which are
manufactured by using open systems, T Cell Product #3 may be
manufactured by using a complete closed system or a semi-closed
system, in which some steps may be performed by using open systems,
e.g., from T cell activation to volume reduction for transduction
and/or from harvest to washing, concentration, and
cryopreservation.
[0308] FIG. 31 shows the turnaround time from leukapheresis
collection to infusion-ready TCR T Cell Product #1 may take about
30 days, e.g., about 14 days from sample collection to harvest and
about 16 days from quality control (QC) to product release; and the
turnaround time for manufacturing TCR T Cell Product #2 may take
about 26 days, e.g., about 10 days from sample collection to
harvest and about 16 days from QC to product release.
[0309] There is, however, a need for fast turnaround. FIG. 30
shows, T Cell Product #3 was manufactured using shorter
manufacturing process, e.g., 5-6 days, from "optional thaw, rest,
and activation" (Step 2) to "harvest and cryopreservation of drug
product cells" (Step 6), using semi-closed system. FIG. 31 shows
TCR T Cell Product #3 may take about 23 days to manufacture, e.g.,
about 7 days from sample collection to harvest and about 16 days
from QC to product release. For commercial manufacturing, for
example, TCR T cell products, e.g., T Cell Product #1, T Cell
Product #2, and T Cell Product #3, may take about 13 days to
manufacture, e.g., about 6 days from sample collection to harvest
and about 7 days from QC to product release.
[0310] FIG. 32 shows a T cell manufacturing process 320 using fresh
PBMCs, which is not obtained by thawing cryopreserved PBMC, thus,
minimizing cell loss due to freezing, thawing, and/or resting PBMCs
and maximizing cell numbers at the beginning of manufacturing
process. For example, T cell manufacturing process 320 may include
Day 0, isolation of fresh PBMC (321), activation of fresh
lymphocytes (322) using, for example, anti-CD3 and anti-CD28
antibodies (soluble or surface bound, e.g., magnetic or
biodegradable beads) in bags, e.g., Saint-Gobain VueLife AC Bags,
coated with anti-CD3 and anti-CD28 antibodies; Day 1, transduction
with CAR or TCR (323) using, for example, lentiviral or retroviral
constructs encoding CAR or TCR or non-viral methods, e.g.,
liposomes; and Day 2, expansion of lymphocytes, Day 5/6, harvest,
and cryopreservation (324) in the presence of cytokine(s), serum
(ABS or FBS), and/or cryopreservation media.
[0311] Improved Product Profile with Shorter Expansion
[0312] The quality, efficacy, longevity, and location of T cell
immunity may result from the diversification of naive T cells
(T.sub.n) into various phenotypically distinct subsets with
specific roles in protective immunity. These include memory stem
(T.sub.scm), central memory (T.sub.cm), effector memory (T.sub.em),
and highly differentiated effector (T.sub.eff) T cells. The
antigen-specific T.sub.n give rise to long-lived T.sub.scm and Tail
that self-renew and provide proliferating populations of
shorter-lived T.sub.em and T.sub.eff cells. Therefore, selecting
less differentiated T.sub.n, T.sub.scm or T.sub.cm subsets for
genetic modification may provide cells with greater therapeutic
efficacy.
[0313] To evaluate the differentiation status of T cell products
harvested at different time of manufacturing, CD8+ T cells obtained
from 3 donors (Donor 1, Donor 2, and Donor 3) were harvested on Day
4 (expansion for 3 days), 7 (expansion for 6 days) and 10
(expansion for 9 days) of manufacturing followed by T cell memory
phenotyping analysis.
[0314] FIG. 33 shows the amount of CD8+ T cells exhibiting the less
differentiated phenotypes, e.g., T.sub.n/scm-CD45RA+CCR7+ and
T.sub.cm-CD45RO+CCR7+, decreases in an expansion time-dependent
manner, i.e., Day 4>Day 7>Day 10. Conversely, the amount of
CD8+ T cells exhibiting the more differentiated phenotypes, e.g.,
T.sub.em-CD45RO+CCR7- and T.sub.eff-CD45RA+CCR7-, increases in an
expansion time-dependent manner, i.e., Day 4<Day 7<Day 10,
indicating more less differentiated phenotypes of Day 4 expanded
cells than that of Day 7 and Day 10 expanded cells. These results
suggest the shorter the T cells expand, the more the T cells
exhibit less differentiated memory phenotypes, thus, with greater
therapeutic efficacy.
[0315] CD27 and CD28 co-stimulation may be required during primary
CD8+ T cell responses. This co-stimulation may provide
proliferation and survival cues to naive CD8+ T cells. To evaluate
the CD27 and CD28 co-stimulation potentials of T cell products
harvested at different time of manufacturing, CD8+ T cells obtained
from 3 donors (Donor 1, Donor 2, and Donor 3) were harvested on Day
4, 7 and 10 of manufacturing followed by CD27 and CD28 expression
analysis.
[0316] FIG. 34 shows the amount of CD8+ T cells exhibiting the
CD27+CD28+ co-stimulation phenotypes decreases in an expansion
time-dependent manner, i.e., Day 4>Day 7>Day 10, indicating
superior CD27 and CD28 co-stimulation of Day 4 expanded cells to
that of Day 7 and Day 10 expanded cells. These results suggest, in
general, the shorter the T cells expand, the more the T cells
express both CD27 and CD28.
[0317] To evaluate the replicative potentials of T cell products
harvested at different time of manufacturing, T cells were
harvested on Day 4, 7 and 10 of manufacturing and monitored for
growth in response to relevant cytokines, e.g., IL-7, IL-15, or
IL-2 in cytokine sensitivity assay.
[0318] FIG. 35 shows T cell growth induced by IL-7, IL-15, or IL-2
for about 21 days decreases in an expansion time-dependent manner,
i.e., Day 4>Day 7>Day 10, indicating superior replicative
potentials of Day 4 expanded cells to that of Day 7 and Day 10
expanded cells. These results suggest the shorter the T cells
expand, the more the T cells respond to cytokines for
proliferation.
[0319] To evaluate the anti-tumor activity of T cell products
harvested at different time of manufacturing, T cell products
obtained from 4 donors (Donor 1, Donor 2, Donor 3, and Donor 4)
were harvested on Day 5, 7 and 9 of manufacturing followed by
interferon-gamma (IFN-.gamma.) release assays in response to
exposure to target positive cell line.
[0320] FIG. 36 shows IFN-.gamma. secretion decreases in an
expansion time-dependent manner, i.e., Day 5>Day 7>Day 9,
indicating, in general, superior anti-tumor activity of Day 5
expanded cells to that of Day 7 and Day 9 expanded cells. These
results suggest, in general, the shorter the T cells expand, the
more the T cells secret IFN-.gamma..
[0321] To further evaluate the cytotoxic activity of T cell
products harvested at different time of manufacturing, EC.sub.50
based on IFN-.gamma. response against T2 cells pulsed with
decreasing concentrations of the cognate peptide was
determined.
[0322] FIG. 37 shows EC.sub.50 increases in an expansion
time-dependent manner, i.e., Day 5>Day 7>Day 9, indicating
superior peptide-specific cytotoxic activity of Day 5 expanded
cells to that of Day 7 and Day 9 expanded cells.
[0323] T Cell Product #3 GMP Manufacturing
[0324] Characterization of Products Manufactured with Final T Cell
Product #3 Process
[0325] FIG. 38 shows expansion metrics. In two Technology Transfer
(TT) manufacturing runs and two Process Qualification (PQ)
manufacturing runs (n=4), an average of 1.3.times.10.sup.10 cells
was harvested with >90% viability following short expansion,
e.g., about 6 days.
[0326] FIG. 39 shows surface expression of T Cell Product #3 TCR
detected by flow cytometry using a TCR specific HLA-dextramer. A
representative FACS plot and combined data (Mean.+-.SD) are shown
from Technology Transfer (TT) and Process Qualification (PQ)
manufacturing runs (n=4) performed using leukapheresis products
from healthy donors.
[0327] FIG. 40 shows T-cell memory phenotype of the final T Cell
Product #3, in which T cells produced by Technology Transfer (TT1,
TT2) and Process Qualification (PQ1, PQ2) manufacturing runs
preserve less differentiated phenotype in donors representing
highly variable memory phenotype of T cell populations in PBMC used
for manufacturing (n=4) (T.sub.n/scm--17.9%, 19.2%, 11.2%, 35.0%
T.sub.cm--23.4%, 15.7%, 0.9%, 2.4% T.sub.em--34.8%, 27.0%, 25.9%,
43% T.sub.eff--23.8%, 38.2%, 62.0%, 16.1% respectively)
[0328] FIG. 41 shows IFN-.gamma. release in response to exposure to
target positive (LVR11KEA) and negative (NT) cell lines. T cells
produced by Technology Transfer (TT) and Process Qualification (PQ)
manufacturing runs show specific cytotoxic activity, e.g.,
IFN-.gamma. release, against the target positive cells. No
IFN-.gamma. release was detected against the negative control
cells.
[0329] FIG. 42 shows EC.sub.50 determination based on IFN-.gamma.
response against target cells pulsed with decreasing concentrations
of the cognate peptide. The results show T cells produced by
Process Qualification (PQ1) manufacturing run exhibit anti-tumor
activity (EC.sub.50=0.3149) comparable to that produced by the
positive control in the assay (EC.sub.50=0.7037).
[0330] FIG. 43 shows a representative figure of cytotoxic potential
of T Cell Product #3 in the Incucyte.RTM. killing assay. Data is
presented as fold tumor growth in the presence of T Cell Product #3
over 72h co-culturing period with a target negative cell line
pulsed with decreasing concentration of the relevant peptide. The
results show a peptide dose dependent killing of target cells by T
cells produced by Process Qualification (PQ1) manufacturing
run.
[0331] In sum, shorter ex-vivo expansion and overall "turnaround
time" can have a substantial impact not only on the quality of the
cell product but also clinical applicability of cellular
immunotherapies. The process development efforts to shorten the
expansion phase during GMP manufacturing of TCR engineered T cells
were completed with the development of a robust, 5-6 day long,
semi-closed T cell manufacturing process for T Cell Product #3. The
Technology Transfer (TT) and Process Qualification (PQ) runs for T
Cell Product #3 manufacturing in GMP environment cleanroom
confirmed the reproducibility and feasibility of the manufacturing
process with shortened expansion phase. All the release, phenotype,
and functionality testing of the TCR engineered T cells were
confirmed for the GMP manufactured T cell products.
Example 7
[0332] Manufacturing and Functionality of T Cell Products Generated
from Cancer Patients
[0333] As noted above, T Cell Product #3 generated from healthy
donors show T-cell memory phenotype and cytotoxic potentials. As
shown below, similar characteristics were observed in T Cell
Product #3 generated from cancer patients, when compared with that
of T Cell Product #3 generated from healthy donors.
[0334] Patient and Donor Characteristics
TABLE-US-00009 Disease Patient Status/Chemo (PT)/Donor Primary
Clinical Treatment Treatment Status: (D) diagnosis Age Gender Race
Stage Status Treatment Notes PT1 Ovarian 78 Female W IV
Stable/Active Cisplatin/Gemzar Cancer Treatment PT2 Ovarian 69
Female W III-C Stable/Active Doxil Cancer Treatment PT3 Ovarian 73
Female W III-B Stable/Active Carboplatin/Gemzar Cancer Treatment
PT4 Endometrial 72 Female AI III-A Unknown/ Taxol/Carboplatin
Cancer Pre-treatment D1 Normal 69 Male W N/A Unknown N/A D2 Normal
70 Male W N/A Unknown N/A D3 Normal 62 Male W N/A Unknown N/A D4
Normal 52 Female H N/A Unknown N/A W = White; AI = American Indian;
H = Hispanic
[0335] T Cell Product #3 were manufactured in small scale using
PBMC obtained from cancer patients and healthy donors. Briefly, on
Day 0, cryopreserved PBMC isolated from leukapheresis products of 4
cancer patients and 4 healthy donors were thawed and rested in the
presence of IL-7 for about 4-6 hours, followed by activation in NTC
24-well plates and incubation for about 16-24 hours. On Day 1,
cells transduced with viral vector expressing recombinant TCR,
e.g., R11KEA TCR, at 5 .mu.l/10.sup.6 cells. Non-transduced (NT))
cells were included as controls. Transduced and non-transduced
cells were seeded at a minimum of 1.0.times.10.sup.6 cells/ml,
e.g., 2.0.times.10.sup.6 cells/ml. On Day 2, Transduced and
non-transduced cells were expanded in TexMACS complete medium with
IL-7 and IL-15. On Day 6, i.e., expansion for 4 days, expanded
cells were harvested followed by flow cytometry analysis and
functional assays to determine, e.g., recovery, viability,
phenotypes, integrated DNA copy numbers, and functionality.
[0336] FIG. 44 shows comparable recoveries of T cells obtained from
cancer patients (Pt) and healthy donors (HD) at post-thawing,
post-resting, and post-activation.
[0337] FIG. 45 shows comparable total viable cells and % viability
of T Cell Product #3 on Day 6, i.e., expansion for 4 days, in
transduced and non-transduced cells within each individual, except
PT1 and PT4, in which all cells were transduced.
[0338] FIG. 46 shows comparable fold-expansion of T Cell Product #3
on Day 6, i.e., expansion for 4 days, in transduced and
non-transduced cells within each individual, except PT1 and PT4, in
which all cells were transduced.
[0339] Phenotype Analysis
[0340] FIG. 47 shows preferential expansion of CD3+CD8+ cells (as
indicated by arrows), as compared with that of CD3+CD4+ cells, in
PBMCs obtained from cancer patients (PT1-PT4) and healthy donors
(D1-D4).
[0341] FIG. 48 shows comparable overall averages of the CD3+CD8+
cells and the CD3+CD4+ cells in T Cell Product #3 and
non-transduced cells (NT) obtained from patients (PT1-PT4) and
healthy donors (D1-D4).
[0342] FIG. 49 shows an example of flow cytometry analysis of T
Cell Product #3. The results indicate 43.8% of T Cell Product #3
contain CD3+CD8+ cells, in which 64.7% of the cells expressing
R11KEA TCR, as indicated by peptide/MHC dextramer (Dex) staining,
and 35.3% of the cells that do not express R11KEA TCR.
[0343] FIG. 50 shows comparable R11KEA TCR expression in CD8+ T
Cell Product #3 generated from cancer patients (PT1-PT4) and
healthy donors (D1-D4).
[0344] FIG. 51 also shows comparable average R11KEA TCR expression
in CD8+ T Cell Product #3 generated from cancer patients (PT1-PT4)
(e.g., 64.3%) and healthy donors (D1-D4) (e.g., 68.2%).
[0345] FIG. 52 shows gating scheme to determine T cell memory
(T.sub.memory) phenotype of T Cell Product #3. For example, by
gating for CD45RA and CCR7, naive "young" T cells (CD45RA+CCR7+),
terminally differentiated "old" T cells (TemRA) (CD45RA+CCR7-),
effector memory T cells (Tem) (CD45RA-CCR7-), and central memory T
cell (Tcm) (CD45RA-CCR7+) can be identified.
[0346] FIG. 53 shows notable average increases in both desirable
naive and Tcm compartments of T Cell Product #3 generated from both
patients (PT1-PT4) and healthy donors (D1-D4). These results
suggest that transduced cells may possess greater ability to
persist after infusion and produce longer lasting responses in
vivo.
[0347] Functional Assays
[0348] To determine the functionality of T Cell Product #3, cells
may be stimulated with relevant peptide (e.g., 1 .mu.g/ml) that
specifically binds R11KEA TCR or irrelevant peptide (e.g., 1
.mu.g/ml), which does not bind R11KEA TCR, as a control.
Stimulation with PMA and ionomycin, which activate all lymphocytes,
serves as positive control; and non-stimulation serves as negative
control. After 2 hours of stimulation, protein transport inhibitors
were added. At 6 hours after stimulation, expression of cytokines
and signalling molecules, e.g., CD107a, IFN-.gamma., TNF-.alpha.,
IL-2, and macrophage inflammatory protein-1-beta (MIP-1.beta.), in
CD3+CD8+ cells were evaluated by intracellular staining (ICS).
[0349] FIG. 54 shows an example of T Cell Product #3, after
stimulation with the relevant peptide (d), the expression levels of
CD107a, IFN-.gamma., TNF-.alpha., IL-2, and MIP-1.beta. in T Cell
Product #3 increase as compared with that of stimulation with the
irrelevant peptide (c). Stimulation with PMA and ionomycin, which
activate all lymphocytes, serves as positive control (b); and
non-stimulation serves as negative control (a).
[0350] FIG. 55 shows polyfunctionality of T Cell Product #3. The
numbers 0, 1, 2, 3, 4, and 5 denote, respectively, the portion of T
Cell Product #3 express none, any one, any two, any three, any
four, and all five of CD107a, IFN-.gamma., TNF-.alpha., IL-2, and
MIP-1.beta.. For example, after stimulation with relevant peptide,
more than 50% of the T Cell Product #3 obtained from healthy donor
(D3) transduced with R11KEA TCR (R11) express at least 2 cytokines
from CD107a, IFN-.gamma., TNF-.alpha., IL-2, and MIP-1.beta., as
compared with that of stimulation with irrelevant peptide, i.e., 0%
of cells express at least 2 cytokines. In contrast, there is no
significant difference in cytokine expression in non-transduced
(NT) cells between stimulation with relevant peptide and irrelevant
peptide. These results show T Cell Product #3 generated from
healthy donors and transduced with R11KEA TCR is polyfunctional.
The positive controls, i.e., T cells stimulated with PMA/ionomycin,
exhibit polyfunctionality with or without TCR transduction. The
negative controls, i.e., T cells without stimulation, exhibit poor
functionality with or without TCR transduction.
[0351] FIG. 56 shows, after stimulation with relevant peptide,
polyfunctionality of the R11KEA TCR+ (CD8+Vb8+) T Cell Product #3
generated from healthy donors, e.g., D3 and D2, and from cancer
patients, e.g., PT1, PT2, and PT3. T cells generated from D1, D4,
and PT4 may not appear polyfunctional as determined by these
functional assays. As shown below, T cells generated from D1, D4,
and PT4, however, still have cytotoxic activity against target
cells.
[0352] FIG. 57 shows IFN-.gamma. release from T Cell Product #3
generated from cancer patients, e.g., PT1-PT4, when these cells
were in contact with a high target cell line, which has about 1,000
copies/cell of the relevant peptide presented on the cell surface,
in E:T ratio-dependent manner, e.g., 10:1>3.3:1>1:1. Note
that T cells generated from PT4, which may not appear
polyfunctional in FIG. 56, also show IFN-.gamma. release in E:T
ratio-dependent manner.
[0353] FIG. 58 shows IFN-.gamma. release from T Cell Product #3
generated from healthy donors, e.g., D1-D4, when these cells were
in contact with a high target cell line, which has about 1,000
copies/cell of the relevant peptide presented on the cell surface
in E:T ratio-dependent manner, e.g., 10:1>3.3:1>1:1. Note
that T cells generated from D1 and D4, which may not appear
polyfunctional in FIG. 56, also show IFN-.gamma. release in E:T
ratio-dependent manner.
[0354] FIG. 59 shows average IFN-.gamma. release from T Cell
Product #3 generated from healthy donors (D1-D4), when these cells
were in contact with cells with different levels of relevant
peptide presented on the cell surface, e.g., high-target cell line
that has about 1,000 copies/cell of the relevant peptide presented
on the cell surface, low-target cell line that has about 50
copies/cell of the relevant peptide presented on the cell surface,
and none-target cell line that does not have the relevant peptide
presented on the cell surface, in peptide presentation
level-dependent manner, i.e.,
high-target>low-target>none-target.
[0355] FIG. 60 shows, similarly, average IFN-.gamma. release from T
Cell Product #3 generated from cancer patients (PT1-PT4), when
these cells were in contact with high-target cell line, low-target
cell line, and none-target cell line, in peptide presentation
level-dependent manner, e.g.,
high-target>low-target>none-target.
[0356] FIG. 61 shows lack of killing activity of T Cell Product #3
generated from healthy donor, e.g., D3, in contact with
target-negative cell line, in which the relevant peptide is not
presented on the cell surface. Briefly, T cells generated from D3
transduced with R11KEA TCR (R11) or without transduction (NT) were
co-cultured with target-negative cell line at E:T ratios of 10:1,
3.3:1, and 1:1. Cell killing activity was measured by using
IncuCyte Killing Assay. These results show no significant
difference in cell killing against target-negative cell line
between T cells with (R11) and without (NT) TCR transduction.
[0357] In contrast, FIG. 62 shows TCR-specific killing activity of
T Cell Product #3 generated from healthy donor, e.g., D3, in
contact with target-positive cell line, in which the relevant
peptide is presented on the cell surface. That is, R11KEA
TCR-expressing T cells kill the target-positive cells in E:T
ratio-dependent manner, e.g., 10:1>3.3:1>1:1. In contrast,
there is no significant difference in cell killing between T cells
without transduction (NT) at different E:T ratios.
[0358] FIGS. 63A-63C show TCR-specific killing activity of T Cell
Product #3 transduced with R11KEA TCR (R11) generated from healthy
donors, e.g., D3 and D4, and from cancer patients, e.g., PT1 and
PT2, in contact with target-positive cell line, in which the
relevant peptide is presented on the cell surface. R11KEA TCR
(R11)-expressing T cells generated from D3, D4, PT1, and PT2 kill
the target-positive cells in E:T ratio-dependent manner, e.g., 10:1
(FIG. 63A)>3.3:1 (FIG. 63B)>1:1 (FIG. 63C). In contrast,
there is no significant difference in cell killing between T cells
without R11KEA TCR transduction (NT) at different E:T ratios.
[0359] In sum, these results show T Cell Product #3 process may
generate T cell products expressing TCR transgene with target
specificity. This process works as well with starting material
obtained from cancer patients as from healthy donors. T Cell
Product #3 process takes shorter time than that for preparing T
Cell Products #1 and #2 and yet generates products with large
numbers of naive and Tcm cells. T Cell Product #3 may be
polyfunctional and secrete IFN-.gamma. in response to
target-positive tumor cell lines. T Cell Product #3 may also
exhibit good effector function in cell line killing assays.
[0360] Advantages of the present disclosure may include autologous
T cell manufacturing processes that may shorten resting time to,
e.g., 4-6 hours, activation time to, e.g., 16-20 hours,
transduction time to, e.g., 24 hours, and expansion phase to, e.g.,
5-7 days, for clinical manufacturing of engineered TCR T cell
products. Critical parameters influencing each step may be
systematically evaluated and may be optimized to yield over 10
billion young, tumor-reactive T cells with a strong ability to
recognize and efficiently kill target expressing tumor cells. In
addition to improving the quality of T cell products, these
optimizations may also result in reducing the cost of manufacturing
by 30%. Further, autologous T cell manufacturing processes of the
present disclosure may be scaled up using flask bound and/or bag
bound anti-CD3 and anti-CD28 antibodies for activating T cells to
yield comparable levels of activation, transducibility, and
expansion and these scale-up processes may be faster than processes
using plate bound antibodies.
[0361] All references cited in this specification are herein
incorporated by reference as though each reference was specifically
and individually indicated to be incorporated by reference. The
citation of any reference is for its disclosure prior to the filing
date and should not be construed as an admission that the present
disclosure is not entitled to antedate such reference by virtue of
prior invention.
[0362] It will be understood that each of the elements described
above, or two or more together may also find a useful application
in other types of methods differing from the type described above.
Without further analysis, the foregoing will so fully reveal the
gist of the present disclosure that others can, by applying current
knowledge, readily adapt it for various applications without
omitting features that, from the standpoint of prior art, fairly
constitute essential characteristics of the generic or specific
aspects of this disclosure set forth in the appended claims. The
foregoing embodiments are presented by way of example only; the
scope of the present disclosure is to be limited only by the
following claims.
Sequence CWU 1
1
157110PRTHomo sapiens 1Tyr Leu Tyr Asp Ser Glu Thr Lys Asn Ala1 5
1029PRTHomo sapiens 2His Leu Met Asp Gln Pro Leu Ser Val1
539PRTHomo sapiens 3Gly Leu Leu Lys Lys Ile Asn Ser Val1 549PRTHomo
sapiens 4Phe Leu Val Asp Gly Ser Ser Ala Leu1 5510PRTHomo sapiens
5Phe Leu Phe Asp Gly Ser Ala Asn Leu Val1 5 1069PRTHomo sapiens
6Phe Leu Tyr Lys Ile Ile Asp Glu Leu1 5711PRTHomo sapiens 7Phe Ile
Leu Asp Ser Ala Glu Thr Thr Thr Leu1 5 1089PRTHomo sapiens 8Ser Val
Asp Val Ser Pro Pro Lys Val1 598PRTHomo sapiens 9Val Ala Asp Lys
Ile His Ser Val1 5109PRTHomo sapiens 10Ile Val Asp Asp Leu Thr Ile
Asn Leu1 5119PRTHomo sapiens 11Gly Leu Leu Glu Glu Leu Val Thr Val1
51210PRTHomo sapiens 12Thr Leu Asp Gly Ala Ala Val Asn Gln Val1 5
101310PRTHomo sapiens 13Ser Val Leu Glu Lys Glu Ile Tyr Ser Ile1 5
10149PRTHomo sapiens 14Leu Leu Asp Pro Lys Thr Ile Phe Leu1
5159PRTHomo sapiens 15Tyr Thr Phe Ser Gly Asp Val Gln Leu1
5169PRTHomo sapiens 16Tyr Leu Met Asp Asp Phe Ser Ser Leu1
5179PRTHomo sapiens 17Lys Val Trp Ser Asp Val Thr Pro Leu1
51811PRTHomo sapiens 18Leu Leu Trp Gly His Pro Arg Val Ala Leu Ala1
5 101911PRTHomo sapiens 19Lys Ile Trp Glu Glu Leu Ser Val Leu Glu
Val1 5 10209PRTHomo sapiens 20Leu Leu Ile Pro Phe Thr Ile Phe Met1
5219PRTHomo sapiens 21Phe Leu Ile Glu Asn Leu Leu Ala Ala1
52211PRTHomo sapiens 22Leu Leu Trp Gly His Pro Arg Val Ala Leu Ala1
5 10239PRTHomo sapiens 23Phe Leu Leu Glu Arg Glu Gln Leu Leu1
5249PRTHomo sapiens 24Ser Leu Ala Glu Thr Ile Phe Ile Val1
5259PRTHomo sapiens 25Thr Leu Leu Glu Gly Ile Ser Arg Ala1
5269PRTHomo sapiens 26Ile Leu Gln Asp Gly Gln Phe Leu Val1
52710PRTHomo sapiens 27Val Ile Phe Glu Gly Glu Pro Met Tyr Leu1 5
10289PRTHomo sapiens 28Ser Leu Phe Glu Ser Leu Glu Tyr Leu1
5299PRTHomo sapiens 29Ser Leu Leu Asn Gln Pro Lys Ala Val1
5309PRTHomo sapiens 30Gly Leu Ala Glu Phe Gln Glu Asn Val1
5319PRTHomo sapiens 31Lys Leu Leu Ala Val Ile His Glu Leu1
5329PRTHomo sapiens 32Thr Leu His Asp Gln Val His Leu Leu1
53311PRTHomo sapiens 33Thr Leu Tyr Asn Pro Glu Arg Thr Ile Thr Val1
5 10349PRTHomo sapiens 34Lys Leu Gln Glu Lys Ile Gln Glu Leu1
53510PRTHomo sapiens 35Ser Val Leu Glu Lys Glu Ile Tyr Ser Ile1 5
103611PRTHomo sapiens 36Arg Val Ile Asp Asp Ser Leu Val Val Gly
Val1 5 10379PRTHomo sapiens 37Val Leu Phe Gly Glu Leu Pro Ala Leu1
5389PRTHomo sapiens 38Gly Leu Val Asp Ile Met Val His Leu1
5399PRTHomo sapiens 39Phe Leu Asn Ala Ile Glu Thr Ala Leu1
5409PRTHomo sapiens 40Ala Leu Leu Gln Ala Leu Met Glu Leu1
5419PRTHomo sapiens 41Ala Leu Ser Ser Ser Gln Ala Glu Val1
54211PRTHomo sapiens 42Ser Leu Ile Thr Gly Gln Asp Leu Leu Ser Val1
5 10439PRTHomo sapiens 43Gln Leu Ile Glu Lys Asn Trp Leu Leu1
5449PRTHomo sapiens 44Leu Leu Asp Pro Lys Thr Ile Phe Leu1
5459PRTHomo sapiens 45Arg Leu His Asp Glu Asn Ile Leu Leu1
5469PRTHomo sapiens 46Tyr Thr Phe Ser Gly Asp Val Gln Leu1
5479PRTHomo sapiens 47Gly Leu Pro Ser Ala Thr Thr Thr Val1
54811PRTHomo sapiens 48Gly Leu Leu Pro Ser Ala Glu Ser Ile Lys Leu1
5 10499PRTHomo sapiens 49Lys Thr Ala Ser Ile Asn Gln Asn Val1
5509PRTHomo sapiens 50Ser Leu Leu Gln His Leu Ile Gly Leu1
5519PRTHomo sapiens 51Tyr Leu Met Asp Asp Phe Ser Ser Leu1
5529PRTHomo sapiens 52Leu Met Tyr Pro Tyr Ile Tyr His Val1
5539PRTHomo sapiens 53Lys Val Trp Ser Asp Val Thr Pro Leu1
55411PRTHomo sapiens 54Leu Leu Trp Gly His Pro Arg Val Ala Leu Ala1
5 10559PRTHomo sapiens 55Val Leu Asp Gly Lys Val Ala Val Val1
5569PRTHomo sapiens 56Gly Leu Leu Gly Lys Val Thr Ser Val1
5579PRTHomo sapiens 57Lys Met Ile Ser Ala Ile Pro Thr Leu1
55811PRTHomo sapiens 58Gly Leu Leu Glu Thr Thr Gly Leu Leu Ala Thr1
5 10599PRTHomo sapiens 59Thr Leu Asn Thr Leu Asp Ile Asn Leu1
5609PRTHomo sapiens 60Val Ile Ile Lys Gly Leu Glu Glu Ile1
5619PRTHomo sapiens 61Tyr Leu Glu Asp Gly Phe Ala Tyr Val1
56211PRTHomo sapiens 62Lys Ile Trp Glu Glu Leu Ser Val Leu Glu Val1
5 10639PRTHomo sapiens 63Leu Leu Ile Pro Phe Thr Ile Phe Met1
56410PRTHomo sapiens 64Ile Ser Leu Asp Glu Val Ala Val Ser Leu1 5
106510PRTHomo sapiens 65Lys Ile Ser Asp Phe Gly Leu Ala Thr Val1 5
106611PRTHomo sapiens 66Lys Leu Ile Gly Asn Ile His Gly Asn Glu
Val1 5 10679PRTHomo sapiens 67Ile Leu Leu Ser Val Leu His Gln Leu1
5689PRTHomo sapiens 68Leu Asp Ser Glu Ala Leu Leu Thr Leu1
56913PRTHomo sapiens 69Val Leu Gln Glu Asn Ser Ser Asp Tyr Gln Ser
Asn Leu1 5 107011PRTHomo sapiens 70His Leu Leu Gly Glu Gly Ala Phe
Ala Gln Val1 5 10719PRTHomo sapiens 71Ser Leu Val Glu Asn Ile His
Val Leu1 5729PRTHomo sapiens 72Tyr Thr Phe Ser Gly Asp Val Gln Leu1
5739PRTHomo sapiens 73Ser Leu Ser Glu Lys Ser Pro Glu Val1
57410PRTHomo sapiens 74Ala Met Phe Pro Asp Thr Ile Pro Arg Val1 5
10759PRTHomo sapiens 75Phe Leu Ile Glu Asn Leu Leu Ala Ala1
5769PRTHomo sapiens 76Phe Thr Ala Glu Phe Leu Glu Lys Val1
5779PRTHomo sapiens 77Ala Leu Tyr Gly Asn Val Gln Gln Val1
5789PRTHomo sapiens 78Leu Phe Gln Ser Arg Ile Ala Gly Val1
57911PRTHomo sapiens 79Ile Leu Ala Glu Glu Pro Ile Tyr Ile Arg Val1
5 10809PRTHomo sapiens 80Phe Leu Leu Glu Arg Glu Gln Leu Leu1
58110PRTHomo sapiens 81Leu Leu Leu Pro Leu Glu Leu Ser Leu Ala1 5
10829PRTHomo sapiens 82Ser Leu Ala Glu Thr Ile Phe Ile Val1
58311PRTHomo sapiens 83Ala Ile Leu Asn Val Asp Glu Lys Asn Gln Val1
5 10849PRTHomo sapiens 84Arg Leu Phe Glu Glu Val Leu Gly Val1
5859PRTHomo sapiens 85Tyr Leu Asp Glu Val Ala Phe Met Leu1
58611PRTHomo sapiens 86Lys Leu Ile Asp Glu Asp Glu Pro Leu Phe Leu1
5 10879PRTHomo sapiens 87Lys Leu Phe Glu Lys Ser Thr Gly Leu1
58811PRTHomo sapiens 88Ser Leu Leu Glu Val Asn Glu Ala Ser Ser Val1
5 108910PRTHomo sapiens 89Gly Val Tyr Asp Gly Arg Glu His Thr Val1
5 109010PRTHomo sapiens 90Gly Leu Tyr Pro Val Thr Leu Val Gly Val1
5 10919PRTHomo sapiens 91Ala Leu Leu Ser Ser Val Ala Glu Ala1
5929PRTHomo sapiens 92Thr Leu Leu Glu Gly Ile Ser Arg Ala1
5939PRTHomo sapiens 93Ser Leu Ile Glu Glu Ser Glu Glu Leu1
5949PRTHomo sapiens 94Ala Leu Tyr Val Gln Ala Pro Thr Val1
59510PRTHomo sapiens 95Lys Leu Ile Tyr Lys Asp Leu Val Ser Val1 5
10969PRTHomo sapiens 96Ile Leu Gln Asp Gly Gln Phe Leu Val1
5979PRTHomo sapiens 97Ser Leu Leu Asp Tyr Glu Val Ser Ile1
5989PRTHomo sapiens 98Leu Leu Gly Asp Ser Ser Phe Phe Leu1
59910PRTHomo sapiens 99Val Ile Phe Glu Gly Glu Pro Met Tyr Leu1 5
101009PRTHomo sapiens 100Ala Leu Ser Tyr Ile Leu Pro Tyr Leu1
51019PRTHomo sapiens 101Phe Leu Phe Val Asp Pro Glu Leu Val1
510211PRTHomo sapiens 102Ser Glu Trp Gly Ser Pro His Ala Ala Val
Pro1 5 101039PRTHomo sapiens 103Ala Leu Ser Glu Leu Glu Arg Val
Leu1 51049PRTHomo sapiens 104Ser Leu Phe Glu Ser Leu Glu Tyr Leu1
51059PRTHomo sapiens 105Lys Val Leu Glu Tyr Val Ile Lys Val1
510610PRTHomo sapiens 106Val Leu Leu Asn Glu Ile Leu Glu Gln Val1 5
101079PRTHomo sapiens 107Ser Leu Leu Asn Gln Pro Lys Ala Val1
51089PRTHomo sapiens 108Lys Met Ser Glu Leu Gln Thr Tyr Val1
510911PRTHomo sapiens 109Ala Leu Leu Glu Gln Thr Gly Asp Met Ser
Leu1 5 1011011PRTHomo sapiens 110Val Ile Ile Lys Gly Leu Glu Glu
Ile Thr Val1 5 101119PRTHomo sapiens 111Lys Gln Phe Glu Gly Thr Val
Glu Ile1 51129PRTHomo sapiens 112Lys Leu Gln Glu Glu Ile Pro Val
Leu1 51139PRTHomo sapiens 113Gly Leu Ala Glu Phe Gln Glu Asn Val1
51149PRTHomo sapiens 114Asn Val Ala Glu Ile Val Ile His Ile1
51159PRTHomo sapiens 115Ala Leu Ala Gly Ile Val Thr Asn Val1
511612PRTHomo sapiens 116Asn Leu Leu Ile Asp Asp Lys Gly Thr Ile
Lys Leu1 5 1011710PRTHomo sapiens 117Val Leu Met Gln Asp Ser Arg
Leu Tyr Leu1 5 101189PRTHomo sapiens 118Lys Val Leu Glu His Val Val
Arg Val1 51199PRTHomo sapiens 119Leu Leu Trp Gly Asn Leu Pro Glu
Ile1 51209PRTHomo sapiens 120Ser Leu Met Glu Lys Asn Gln Ser Leu1
51219PRTHomo sapiens 121Lys Leu Leu Ala Val Ile His Glu Leu1
512210PRTHomo sapiens 122Ala Leu Gly Asp Lys Phe Leu Leu Arg Val1 5
1012311PRTHomo sapiens 123Phe Leu Met Lys Asn Ser Asp Leu Tyr Gly
Ala1 5 1012410PRTHomo sapiens 124Lys Leu Ile Asp His Gln Gly Leu
Tyr Leu1 5 1012512PRTHomo sapiens 125Gly Pro Gly Ile Phe Pro Pro
Pro Pro Pro Gln Pro1 5 101269PRTHomo sapiens 126Ala Leu Asn Glu Ser
Leu Val Glu Cys1 51279PRTHomo sapiens 127Gly Leu Ala Ala Leu Ala
Val His Leu1 51289PRTHomo sapiens 128Leu Leu Leu Glu Ala Val Trp
His Leu1 51299PRTHomo sapiens 129Ser Ile Ile Glu Tyr Leu Pro Thr
Leu1 51309PRTHomo sapiens 130Thr Leu His Asp Gln Val His Leu Leu1
51319PRTHomo sapiens 131Ser Leu Leu Met Trp Ile Thr Gln Cys1
513211PRTHomo sapiens 132Phe Leu Leu Asp Lys Pro Gln Asp Leu Ser
Ile1 5 1013310PRTHomo sapiens 133Tyr Leu Leu Asp Met Pro Leu Trp
Tyr Leu1 5 101349PRTHomo sapiens 134Gly Leu Leu Asp Cys Pro Ile Phe
Leu1 51359PRTHomo sapiens 135Val Leu Ile Glu Tyr Asn Phe Ser Ile1
513611PRTHomo sapiens 136Thr Leu Tyr Asn Pro Glu Arg Thr Ile Thr
Val1 5 101379PRTHomo sapiens 137Ala Val Pro Pro Pro Pro Ser Ser
Val1 51389PRTHomo sapiens 138Lys Leu Gln Glu Glu Leu Asn Lys Val1
513911PRTHomo sapiens 139Lys Leu Met Asp Pro Gly Ser Leu Pro Pro
Leu1 5 101409PRTHomo sapiens 140Ala Leu Ile Val Ser Leu Pro Tyr
Leu1 51419PRTHomo sapiens 141Phe Leu Leu Asp Gly Ser Ala Asn Val1
514210PRTHomo sapiens 142Ala Leu Asp Pro Ser Gly Asn Gln Leu Ile1 5
101439PRTHomo sapiens 143Ile Leu Ile Lys His Leu Val Lys Val1
51449PRTHomo sapiens 144Val Leu Leu Asp Thr Ile Leu Gln Leu1
51459PRTHomo sapiens 145His Leu Ile Ala Glu Ile His Thr Ala1
51469PRTHomo sapiens 146Ser Met Asn Gly Gly Val Phe Ala Val1
51479PRTHomo sapiens 147Met Leu Ala Glu Lys Leu Leu Gln Ala1
51489PRTHomo sapiens 148Tyr Met Leu Asp Ile Phe His Glu Val1
514911PRTHomo sapiens 149Ala Leu Trp Leu Pro Thr Asp Ser Ala Thr
Val1 5 101509PRTHomo sapiens 150Gly Leu Ala Ser Arg Ile Leu Asp
Ala1 51519PRTHomo sapiens 151Ser Tyr Val Lys Val Leu His His Leu1
51529PRTHomo sapiens 152Val Tyr Leu Pro Lys Ile Pro Ser Trp1
51539PRTHomo sapiens 153Asn Tyr Glu Asp His Phe Pro Leu Leu1
51549PRTHomo sapiens 154Val Tyr Ile Ala Glu Leu Glu Lys Ile1
515512PRTHomo sapiens 155Val His Phe Glu Asp Thr Gly Lys Thr Leu
Leu Phe1 5 101569PRTHomo sapiens 156Val Leu Ser Pro Phe Ile Leu Thr
Leu1 51579PRTHomo sapiens 157His Leu Leu Glu Gly Ser Val Gly Val1
5
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