Poster
A multipronged approach to improve gene delivery efficiency for in vivo CAR-T using CD3 and CD7 retargeted LVVs
A multipronged approach to improve gene delivery efficiency for
in vivo CAR-T using CD3 and CD7 retargeted LVVs
Abstract Presentation Number: 147
Longshan Liu, Cheng Luo, Lianqu Li, Yangyang Tang, Mangmang Li, Yu Liang
Introduction
Complementary Approaches to Enhance
Transduction
Enhanced T Cell Transduction via Dual CD7-CD3
Targeting
Chimeric antigen receptor (CAR)-T therapies have advanced cancer
treatment; however, for conventional, ex vivo CAR-T therapies, patient access
is often hindered by high costs, long manufacturing times, lymphodepleting
chemotherapy needed to precondition the patient, and the inconvenience to
travel to specialized treatment centers. To address these limitations,
strategies have recently emerged to generate CAR-T cells in situ. These
approaches, comprising the field of in vivo CAR-T, are based on delivering
the CAR construct to T cells in situ typically using either lentiviral vectors
(LVV) or lipid nanoparticles (LNP) decorated with T cell-specific antibodies.
In vivo CAR-T approaches have demonstrated encouraging efficacy in
preclinical and early clinical studies, but still face challenges in terms of
efficiency, specificity, durability, and scalability.
Specific transduction of hu PBMC
using CD7 retargeted LVV
Specific transduction of hu PBMC
using CD3 retargeted LVV
A
CD7 Expression in PBMCs
Transduction Efficiency in PBMCs
PBMC
PBMC + LVV
30
25
20
B
C
Day 5
MOI 5
MOI 20
MOI 80
Day 3
Day 7
8000
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20
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0
CVD A
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CVD A+100 IU IL7
100 IU IL7
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CD3 and CD7 are well-established T cell targets used in current in vivo CAR-
T delivery vehicles, and such LVV and LNP-based approaches are showing
encouraging efficacy in CAR gene delivery to T cells in situ; however, the
efficiency of current approaches based on monospecific CD3 or CD7
antibodies for in vivo gene delivery still remains low, thereby limiting their
therapeutic potency. By using internally developed CD3 and CD7 VHH
antibodies, we implemented a multipronged approach to improve the gene
delivery efficiency in vivo, including i) incorporation of a CD3+CD7 bispecific
antibody in the LVV particle; ii) integration of a mutated envelope protein that
reduces LDLR-mediated membrane fusion; iii) the inclusion of a HIV
component in the LVV particles that counteracts host antiviral defense
mechanisms to enhance transgene expression; and 4) co-administration of an
approved cardiovascular drug that enhances CD7 expression in T cells.
C
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Nalm-6/Luc
Day -3
1E7 PBMC
Day -1
LVV
CD3 and CD7 VHHs demonstrate strong specificity in retargeting LVV for T
cell transduction. (A) Jurkat cells were transduced with either a CD3-
targeted (left) or CD7-targeted (right) LVV and assayed for transduction
efficiency and specificity using the respective knockout mutants. (B&C)
human PBMCs were transduced with a GFP-encoding LVV retargeted by CD3
VHH (B) or CD7 VHH (C), and assayed for transduction in target-positive (top
row) and target-negative (bottom row) cells via flow cytometry at the
indicated times/MOIs.
NOG
MHC I/II KO
Day 0
40
CD7 LVV
35
30
CD7 LVV w/ HIV-vif
10
In Vivo CAR Expression
25
20
15
PBMC
5
1E7 TU
4
3
2
1
0
CD7-CD3 T Cell
Engineered De-
1E7 TU + IL-7 (200 ng)
1E7 TU + CVD A
Targeting + Activation
targeted Envelope
5
0
Complex
Specifically targets
LVV to T cells and
Protein
A
Transduction Efficiency
T Cell Activation
Reduces
interaction of LVV
induces their robust
with LDLR to
3
5
7
activation resulting in
mitigate non-T cell
greater transduction
transduction
Days post-transduction
efficiency
7
14
21
Days Post-LVV Administration
Approved cardiovascular drug (CVD A) and HIV-Vif incorporation augments
LVV transduction efficiency. (A) PBMCs or PBMCs + a GFP-encoding LVV
were co-administered with the indicated test article and CD7 expression was
assessed via flow cytometry at Day 3 (left) while transduction efficiency was
assessed at the indicated time points (right). (B) CAR expression was
assessed in a rodent PBMC model following intravenous administration of a
CD19 CAR-encoding LVV and the indicated test article. (C) Jurkat cells were
transduced with a CD7-targeted LVV ± HIV-Vif and assessed for transduction
efficiency via GFP expression.
HIV-based Host
Defense
Suppressor
Co-administration
with Cardiovascular
Drug
Counteracts the
Approved, small
host cells’ anti-viral
molecule drug
defense
mechanisms to
improve expression
of GOI
increases T cell
expression of CD7
and enhances viral
transduction
B
CAR-T-Mediated B Cell Depletion
100
80
60
40
20
0
LVV
i.v.
Vehicle
µ
CD3 (5 L)
B cell depletion, assessed
µ
CD3 (20 L)
weekly
Enhanced T Cell Transduction via Dual CD7-CD3
Targeting
µ
CD7 (5 L)
Summary
huCD34+ model
12 weeks
Day 0
µ
CD7 (20 L)
µ
CD7-CD3 (5 L)
µ
CD7-CD3 (20 L)
We have developed a multipronged approach to improve LVV transduction
efficiency to T cells in vivo. Our anti-CD7-CD3 VHH fusion moiety
specifically targets LVVs to T cells and the dual binding synergistically
improves transduction efficiency over CD3- or CD7-targeting alone. Co-
administration of an approved, small molecule cardiovascular drug, which
increases expression of CD7 in T cells, can complement our CD7-CD3-
targeted LVV to further enhance transduction efficiency. Moreover,
incorporation of the HIV-Vif into the viral particle effectively suppresses
host anti-viral defenses to enhance transgene expression. These
technologies provide for the creation of an in vivo TCR/CAR-T delivery
platform with enhanced efficiency.
Specific Transduction of T cell lines
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
Days post-treatment
100
80
60
40
20
0
100
80
60
40
20
0
A
Jurkat CD3KO
Jurkat
Jurkat CD7KO
Jurkat
Retargeting LVV by CD7-CD3 bispecific antibody enhances T cell
transduction and activation, resulting in greater in vivo target cytotoxicity.
(A) human PBMC was transduced with a variety of Ab retargeted LVVs,
including monospecific CD7 or CD3 VHH or CD7xCD3 bispecific antibodies,
and assayed for transduction efficiency (left) and T cell activation (right). (B)
CD34-humanized mice were injected intravenously with a CD19 CAR-
encoding LVV with the indicated targeting moiety and B cell depletion was
assessed via blood draw at the indicated time points.
100
10
1
0.1
0.01
0.001
256 128
64
32
16
8
4
Volume (μL)
Volume (μL)
ProBioCDMO.com
Contacts: Yu Liang, Ph.D.; Vice President of Discovery, yu.liang@probiocdmo.com; Suraj Kachgal, Ph.D.; Head of Global Asset & Technology Licensing, suraj.kachgal@genscript.com