Using single B platform to develop a CAR T-celltherapeutic solution for a GPCR target

Kaikai Zeng*, Antao He*, Shujuan Hong*, Fen Mei*, Meng Cai*, Chong Dai*, Min Luo#, Guangchao Li#, Donghui Ma**DIMA Biotech Ltd, Wuhan institute of Biotechnology B7, Biolake No.666 Gaoxin Road, Wuhan, Hubei, China,# Guangzhou Bio-gene Technology Co., Ltd, 206 Kaiyuan Blvd, Huangpu Qu, Guangzhou City, Guangdong Province, China

Abstract

Experimental approach

CHTo expedite antibody-based drug development, DIMA Biotech developed more than 5000 on-shelf lead antibody molecules on over
300 druggable targets with its DimAb® single B platform. In this presentation, we will elaborate DimAb® platform and showcase how
we developed anti-GPRC5D CAR T-cell therapy constructs from leads to IIT clinical trial stage with our collaborator. The preliminary
data from ongoing clinical trial exhibits astonishing clinical efficacy on RRMM patients.

Figure 1. DiMProTM multi-passtransmembrane protein
production and purification
platform. DIMA biotech has
established a plethora oftechnology platforms toproduce functional membraneproteins as immunogen fortherapeutic antibody
development.

BIOTE

DiMProTM membrane protein purification platform

BDIMAADimAb® single B cell platform

AB

Figure 2. (A) DimAb® single B cell workflow. DimAb®
single B platform takes advantages of unique B cell
culture method, which allows fast and precise
isolation of positive antibody sequences. (B) DimAb®
B cell library. The unused B cells from immunizedanimals can be stored in liquid nitrogen for longterm. More lead antibody molecules can be selectedby initiating a new round of screening and cloning,which takes only 30 days.

DiLibraryTM antibody humanization platform

Figure 3. (A) The workflow of DiLibraryTM antibodyhumanization. CDRs from parental antibody are first
grafted onto a FR library constructed from a numberof selected human IgG germline sequences. The final
humanized antibody library will be displayed on
HEK293 cell surface, and the best binders can beselected via FACS sorting with target protein. (B)Examples of two humanization experiments. The
selected humanized antibodies show improved
affinities (pM) comparing to the parental antibody.

ResultsA

B

Anti GPRC5D scFv hFc

CHAnti GPRC5D scFv hFc

Figure 4. Affinity ranking of 9 humanized anti-GPRC5D DimAbs through flow assay. Comparing to BMK antibody (G00)
pointed by red arrow, derived from a CAR construct currently in phase I clinical trial, 4 out of 9 DimAbs exhibit stronger binding
affinity on both RPMI8226 or K562-GPRC5D stable cell line.

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CAR-T-GPRC5D / 8226-Luc 24 hours of co-culture

DIMA

A

Figure 6. The selected CAR constructs exhibit highspecificity in activation upon contact of target cells. We
examined the specificity in activation of G00, G05, G07, andG09 CAR constructs through a CAR-Jurkat activation assay,and all CAR constructs show exclusive target cell specificity.

BI

Figure 5. In Vitro tumor cell cytotoxicity assay. The
experimental data showed that several of CARs constructedfrom DimAbs exhibit stronger cytotoxic capacity thanbench-mark CAR (G00), pointed by red arrows, especially
under low Effector:Tumor ratio.

BMM tumor before and after CAR T-cell treatment

BeforeAfterFigure 7. Animal survival data and early IIT clinical trial data. A. Multiple myeloma (MM) model mice infused with two dosageof CAR T-cells exhibit elongated survival time comparing with control groups. B. A RRMM patient relapsed from BCMA CAR T-celltherapy was treated with our anti-GPRC5D CAR T-cells. A metastatic tumor of large size behind patient ear nearly disappeared in
10 days after CAR T-cells infusion. (Currently our collaborators are recruiting more patients.)

Conclusion

DIMA Biotechnology Ltd is a biotech company dedicated on hit to lead stage therapeutic antibody discovery. Currently we haveobtained more than 5000 IgG molecules on over 300 targets. In this anti-GPRC5D CAR T-cell project, we showcased how wedeveloped 3 lead stage DimAbs and then chose one to push into IIT clinical trial. For this project, our collaborator is planning torecruit 18 patients for IIT and the clinical data will be reported separately.