Poster
Accelerating CAR-T Antibody Discovery
Utilization of naïve VHH phage display library & integrated
antibody engineering platform to generate antibody leads
for CAR-T therapy
Li Chen, Yu Liang, Yingyu Li, Wenwan Fang, Jie Ma
ProBio USA Inc
ABSTRACT
2.VHH candidates against three different ECD domains were
Due to its small size and stable, single-domain nature VHHs or single-domain antibody (sdAb) has become
obtained by differential panning/screening strategy
an ideal building block in cell therapy and achieved encouraging success clinically, such as in the case of
CAR-T therapy based on anti-BCMA VHH (CARVYKTI® by Legend/J&J). However, VHH discovery based on
the conventional immunized phage library is sometimes hampered by the poor sequence/epitope diversity
and low immune response due the high AA sequence homology of the target protein between human
lg lg-like domain
and the host species. To address these limitations, here in this report, we present an alternative approach
AHP15485
utilizing an alpaca naïve VHH library combined with antibody engineering by affinity maturation.
EMB-07
Frizzled domain
FZD
AHP15662
AHP15580
NVG-111
INTRODUCTION
In this case study, an alpaca naïve VHH library was panned and screened by ROR1 proteins of 3 different
ECDs and CHO-K1/ROR1 cell line to identify ROR1 VHH candidates. These VHH candidates were used to construct
CAR-T cells and further evaluated by vitro functional assays, based on which final leads were selected for
optimization by affinity maturation and ready for the development of CAR-T therapies.
KRD Kringle domain
MATERIALS AND METHODS
Figure 2 The naïve library gives a panel of diversified candidates, binding to three different domains
:
of ROR1 extra-cellular region.
1.Generation of naïve phage display library
3.Functional evaluation of ROR1 VHH leads in a CAR-T setting
PBMCs from hundreds of naïve alpaca donors were collected and used to build a naïve VHH phage library
11
with the size of 2X10 .
Comparing with immunized
library, whose success is often
limited by poor epitope & sequence
diversity and low immune response
when the AA sequence homology
of the target is high between
A
Luciferase Based Killing Assay
VHH
h
CH2
VHH
hu Ag and alpaca, such as in
the case of ROR1, the naïve library
may offer better epitope/sequence
diversity and faster turnaround
time.
VHH
SfiI
VHH
Figure 3 Functional evaluation of ROR1 VHHs in a
:
SfiI
CAR-T setting in vitro.
CAR-T cells was constructed by transduction of
primary T cells with CAR constructs containing
relevant VHH candidates, and evaluated by in
vitro function assays.
2.Phage library panning & screening
To obtain VHH candidates recognizing different ECDs, the naïve phage library was panned and
screened by three different ECD proteins and ROR1 overexpressing cell line, followed by phage monoclonal
screening via ELISA, SPR and FACS.
(A) Cytotoxicity assay was performed after co-in-
cubation of CAR-T cells (effectors) and
CHO-K1/ROR1/Luc for 24 hours. The percentage of
target cell lysis was calculated per luminescence
signal.
ROR1 CAR T cell activation assay_IFNγ production
B
Geometric Mean：Alexa Fluor 647-H
3097
75.0
(B) The release of representative cytokines
(IFN-γ) by ROR1 CAR-T cells in response to
co-incubation with target cells was measured by
ELISA assays.
50
0
0
250
Alexa Fluor 647-H
Monoclonal FACS
Monoclonal ELISA
SPR Ranking
VHH
CD28 TM
3.CAR-T cell construction and
functional evaluation
4-1BB
VHH candidates were used to build CAR domain construct via
transduction of T-cells to make CAR-T cells, and further evaluated
in cell killing and cytokine release assays in vitro.
CD3 ζ
4.VHH lead optimization by humanization & affinity maturation
T2A
(A) Parental VHH
KD= 280nM
(B) Humanized VHH
KD= 320nM
(C) Affinity matured VHH
KD= 8.9nM
RESULTS
1.Library panning & screening funnel
Alpaca naïve library panning by ROR1 ECD
protein (full length, FZD or KRD domain)
Figure 4 Full kinetic SPR analysis revealed that the affinity of a top candidate (AHP15485) was maintained
:
Binding specificity via ELISA/FACS
>400 hits
after humanization, and greatly improved after affinity maturation (> 30 fold).
Sequencing and confirmation
30 ROR1 binder
by IgG conversion
23 ROR1 VHHs
8 ROR1 VHHs
VHH-Fc: ELISA/FACS/bioassay
CONCLUSION
In vitro bioassay: CAR-T
In this report, to address some of the limitations of the conventional VHH lead discovery based on the immunized
library, an alternative approach was presented, combining the advantages of alpaca naïve VHH library and Ab
engineering by affinity maturation. We showed this approach successfully delivered multiple VHH leads
targeting diverse ECD domains of ROR1, a challenging target for an immunized library approach due to its
high AA sequence homology between human and alpaca. These VHH candidates showed promising characteristics
in vitro and are ready for the development of CAR-T therapies.
Humanization &
3 ROR1 VHHs
Affinity maturation
Figure 1 The alpaca naïve VHH library was panned & screened by three ROR1 ECD proteins and CHO-K1/ROR1 stable cell line,
:
followed by functional evaluation in a CAR-T setting. The top three candidates were selected for antibody optimization by
humanization and affinity maturation as final leads.
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