635 A novel site-directed chemical conjugation technology confers antitumor activity via native Fc receptor to plasma immunoglobulin by attaching tumor binders

Background

We describe KPMW101, which was created by chemical conjugation of a CD38-specific binder to clinical grade intravenous immunoglobulin (IvIg) pooled from healthy donors. Kleo's MATE^TM technology enables efficient site-directed chemical conjugation to 'off-the-shelf' IvIg and allows the development of antitumor agents with rapidly introduced target specificity. Our platform allows for chemical engineering of existing IvIg in a cost-efficient manner. This technology relies on synthetic compounds that consists of antibody binder with react-and-release mechanism.

Methods

Design of synthetic chemical reagents included antibody binding group capable of covalent bond formation with specific lysine, CD38 binding moiety proven to work in our clinical candidate KP1237, and tunable non-cleavable linker. Conjugation efficiency to polyclonal IvIg was evaluated using LC-MS analysis of IdeZ-digests. The binding of CD38, CD16a, and FcRn were determined by ELISA and BLI.For in vitro ADCC assays, PBMCs provided NK effector function. Daudi (CD38⁺) B lymphoblast cells were treated with KPMW101 or IvIg, PBMCs were introduced and incubated for 18h, and target cellular death was measured. For an in vivo IP macrophage lavage model of ADCP, SCID mice were implanted IP with CFSE-labeled Daudi cells. Mice were injected with IvIg or KPMW101 (0.21, 0.625, 1.875 mg/kg) SQ, and tumor cell counts were measured by flow cytometry. The pharmacokinetic profile of in vivo KPMW101 was determined from blood and analyzed utilizing a human Ig isotyping array.

Results

Synthetic chemical reagents with multiple linker types have been conjugated to IvIg and evaluated in biochemical assays. KPMW101 showed the highest conjugation efficiency. Binding affinity of KPMW101 to CD38 was 27nM. ELISA results show KPMW101 binds to CD16a and FcRn, indicating that conjugation does not interfere with FcR binding.In vitro ADCC results demonstrate that KPMW101 elicited CD38⁺ target cell killing with an EC₅₀ of 0.91–2.09nM.In vivo studies showed that KPMW101 resulted in a 49.9–63.5% reduction of tumor cells. Pharmacokinetic profile showed stability of KPMW101 throughout the 144-hour study, whereby IgG1, IgG2, IgG3, and IgG4 isotypes were detectable.

Conclusions

KPMW101 is created by chemical conjugation of CD38-specific binder to IvIg using our proprietary MATE^TM technology, maintaining native binding to FcRs via the Fc domain. This ensures the stability of the molecule and retains immune-mediated mechanisms of action. KPMW101 induces IvIg to adopt Fc effector mechanisms like ADCC and ADCP. Our in vitro data and in vivo studies confirm KPMW101 ability to kill tumor cells, making IvIg into an active antitumor therapeutic agent.