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Updated: Jul 8, 2025
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
Development and characterization of NILK-2301, a novel CEACAM5xCD3 κλ bispecific antibody for immunotherapy of
Anja Seckinger1, Sara Majocchi2, Valéry Moine2
1LamKap Bio Alpha AG, Bahnhofstrasse 1, 8808, Pfäffikon, SZ, Switzerland.
Background:
T-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA).
Methods:
We present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format).
Results:
NILK-2301 binds CD3ɛ on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the "LALAPA" mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 (Emax = 89%), MKN-45 (Emax = 84%), and H2122 (Emax = 97%), with EC50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice.
Conclusions:
In summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.
Insights
A novel bispecific antibody, NILK-2301, shows promising preclinical activity against CEACAM5-expressing cancers by enhancing T-cell activation. This antibody format may reduce anti-drug antibody formation and is advancing to clinical trials.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- T-cell retargeting via CEACAM5xCD3 bispecific antibodies (BsAbs) has shown limited clinical success.
- Challenges include insufficient T-cell activation, dose-limiting toxicities, and anti-drug antibody (ADA) formation.
Purpose of the Study:
- To develop and characterize NILK-2301, a novel bispecific antibody (BsAb) for targeting CEACAM5-expressing tumors.
- To evaluate its preclinical efficacy, safety, and pharmacokinetic profile.
Main Methods:
- Generation of NILK-2301, a κλ body format BsAb targeting CD3ɛ and CEACAM5.
- In vitro assessment of T-cell activation, cytotoxicity, and binding affinities.
- In vivo studies in tumor models and pharmacokinetic/tolerability studies in non-human primates.
Main Results:
- NILK-2301 demonstrated potent T-cell activation, proliferation, and dose-dependent cytotoxicity against CEACAM5-positive cancer cell lines.
- Effective tumor growth delay observed in colon and pancreatic cancer models.
- Favorable pharmacokinetics with a projected human half-life of 13.1 days and good tolerability in cynomolgus monkeys.
Conclusions:
- NILK-2301 exhibits significant preclinical efficacy and a favorable safety profile.
- Its unique format may reduce ADA generation, positioning it for clinical development.
- Clinical trials are anticipated to commence by the end of 2023.
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