PharmaceuticalOutsourcingQ42020 - 43

ANALYTICAL TESTING

complicates assay development.18 These complications may require
extensive prescreening of negative individuals for cut point determination and/or negative control pool preparation.
The second piece of B-cell mediated immunogenicity for CGT
concerns antibodies against the expressed protein. Patients in CGT
trials may be heavily pretreated, particularly in cases where there
is an approved enzyme or protein replacement product on the
market. Immunogenicity needs to be evaluated at enrollment and
throughout the study. Populations with a high occurrence of preexisting antibodies may need alternative statistical strategies to
detect and characterize treatment-emergent immunogenicity such
as determining fold-change in ADA titer after treatment.19

of bioanalytical challenges, including assay sensitivity, dynamic
range, analyte stability, pre-existing antibodies, performance of
endogenous vs. recombinant material, cell product and vector (viral
or LNP) heterogeneity, and the need to integrate multiple analytical
platforms. Therefore, early assessment of analytical needs, as well
as technical capabilities, is important to ensure that a bioanalytical
package is produced that can accurately characterize the therapeutic
product's exposure, mechanism of action, efficacy and safety.

References
1. Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M. Gene therapy comes of age.
Science (80- ). 2018;359(6372). doi:10.1126/science.aan4672

The analytical approach to protein immunogenicity usually consists
of standard immunoassay bridging or sandwich assay formats. Some
challenges to method development include obtaining adequate
quantities of quality protein stocks, labeling non-antibody proteins
and obtaining sufficiently sensitive positive control antibodies.
Additionally, protein interference and protein binding partner
interference may be more difficult to engineer out of the assay, so
nontraditional methods of dissociation or target depletion may need
to be employed, such as heat treatment or immunoprecipitation
sample treatments.20

2. Waldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic
science to clinical practice. Nat Rev Immunol. 2020. doi:10.1038/s41577-020-0306-5

ELISPOT (Enzyme Linked Immunospot) assays, monitoring T-cell
immune response, detect specific cytokines or antigen-specific
antibodies as well as the frequency of secreting cells that occur at
low frequencies and can best be detected using this sensitive assay
technique. This assay can detect cytokine or effector molecule
secretion at the single cell level and is more sensitive than the ELISA or
intra-cellular staining techniques. While it requires isolation of PBMCs
or other cell subsets, it is able to be automated, allowing for high
throughput screening. This becomes useful in gene therapy when,
for example, an AAV-based gene transfer anti-capsid T cell response
could eliminate transduced cells and would need to be monitored.21

7. Eyquem J, Mansilla-Soto J, Giavridis T, et al. Targeting a CAR to the TRAC locus with
CRISPR/Cas9 enhances tumour rejection. Nature. 2017;543(7643):113-117. doi:10.1038/
nature21405

Flow cytometry monitors the cellular kinetics of infused cells in
adoptive cell therapy, such as in CAR-T cells. This is important
for evaluating in vivo post antigen exposure-related expansion
and persistence of the infused cells. Flow cytometry can also
immunophenotype panels of multiple cell types often monitored
in cell therapies. One such example is the T-cell lymphocyte
phenotypic panel, which can monitor regulatory markers,
activation, memory or proliferation.
In the case of cell therapies, immunogenicity assessments consider
the presence of ADAs, both pre-existing and treatment-emergent,
against key expressed proteins on the surface of the CAR-T cells. In
designing a bioanalytical approach, research teams must decide on
the specific target proteins on the CAR-T cell for ADA development
and consider whether expressed, soluble protein or cell-linked
protein will be used for assay development and validation.

3. Torikai H, Reik A, Liu PQ, et al. A foundation for universal T-cell based immunotherapy:
T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate
expression of endogenous TCR. Blood. 2012;119(24):5697-5705. doi:10.1182/
blood-2012-01-405365
4. Rezvani K. Adoptive cell therapy using engineered natural killer cells. Bone Marrow
Transplant. 2019;54:785-788. doi:10.1038/s41409-019-0601-6
5. Pipe S, Leebeek FWG, Ferreira V, Sawyer EK, Pasi J. Clinical Considerations for Capsid Choice
in the Development of Liver-Targeted AAV-Based Gene Transfer. Mol Ther - Methods Clin
Dev. 2019;15(December):170-178. doi:10.1016/j.omtm.2019.08.015
6. Acharya UH, Dhawale T, Yun S, et al. Management of cytokine release syndrome and
neurotoxicity in chimeric antigen receptor (CAR) T cell therapy. Expert Rev Hematol.
2019;12(3):195-205. doi:10.1080/17474086.2019.1585238

8. Li C, Mei H, Hu Y. Applications and explorations of CRISPR/Cas9 in CAR T-cell therapy. Brief
Funct Genomics. 2020;19(3):175-182. doi:10.1093/bfgp/elz042
9. Desai AK, Li C, Rosenberg AS, Kishnani PS. Immunological challenges and approaches to
immunomodulation in Pompe disease: a literature review. Ann Transl Med. 2019;7(13):285285. doi:10.21037/atm.2019.05.27
10. Micheli JE, Myler H, Stinchcomb M, Kernstock R. Strategies for Regulated Cell and Genetherapy Based Enzyme Activity Assays. In: PharmSci360. ; 2020.
11. Fehse B, Badbaran A, Berger C, et al. Digital PCR Assays for Precise Quantification of CD19CAR-T Cells after Treatment with Axicabtagene Ciloleucel. Mol Ther - Methods Clin Dev.
2020;16(March):172-178. doi:10.1016/j.omtm.2019.12.018
12. Transl J, Lu A, Liu H, et al. Application of droplet digital PCR for the detection of vector copy
number in clinical CAR / TCR T cell products. J Transl Med. 2020:1-7. doi:10.1186/s12967020-02358-0
13. Kakkanaiah VN, Lang KR, Bennett PK. Flow cytometry in cell-based pharmacokinetics or
cellular kinetics in adoptive cell therapy. Bioanalysis. 2018;10(18):1457-1459. doi:10.4155/
bio-2018-0203
14. Ye G, Budzynski E, Sonnentag P, et al. Safety and Biodistribution Evaluation in CNGB3Deficient Mice of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of
Achromatopsia. Hum Gene Ther Clin Dev. 2016;27(1):27-36. doi:10.1089/humc.2015.163
15. Wang W, Fasolino M, Cattau B, et al. Joint profiling of chromatin accessibility and CAR-T
integration site analysis at population and single-cell levels. Proc Natl Acad Sci U S A.
2020;117(10):5442-5452. doi:10.1073/pnas.1919259117
16. Marco L. Davila1, Isabelle Riviere1, 2, 3, 4, Xiuyan Wang4, Shirley Bartido4, Jae Park1, Kevin
Curran5, Stephen S. Chung1, Jolanta Stefanski4, Oriana Borquez-Ojeda4, Malgorzata
Olszewska4, Jinrong Qu4, Teresa Wasielewska4, Qing He4, Mitsu Fink4, Himaly Sh
3. C-reactive protein may indicate risk of car T cell-induced toxicity. Cancer Discov.
2014;4(4):388. doi:10.1158/2159-8290.CD-RW2014-052
17. Calcedo R, Vandenberghe LH, Gao G, Lin J, Wilson JM. Worldwide Epidemiology
of Neutralizing Antibodies to Adeno-Associated Viruses. 2009;199:381-390.
doi:10.1086/595830
18. Vandamme C, Adjali O, Mingozzi F. Unraveling the Complex Story of Immune Responses
to AAV Vectors Trial After Trial. Hum Gene Ther. 2017;28(11):1061-1074. doi:10.1089/
hum.2017.150
19. Gorovits B, Clements-Egan A, Birchler M, et al. Pre-existing Antibody: Biotherapeutic
Modality-Based Review. AAPS J. 2016;18(2):311-320. doi:10.1208/s12248-016-9878-1

Summary
While many commonalities are present in assay types between
CGT programs and other large-molecule bioanalysis, the distinct
features of cell and gene therapies require attention to a variety
pharmoutsourcing.com |

20. Butterfield AM, Chain JS, Ackermann BL, Konrad RJ. Comparison of assay formats for drugtolerant immunogenicity testing. Bioanalysis. 2010;2(12):1961-1969.
21. Martino AT, Herzog RW, Anegon I, Adjali O. Measuring immune responses to recombinant
AAV gene transfer. Methods Mol Biol. 2011;807:259-272. doi:10.1007/978-1-61779-3707_11

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