Antibody–drug conjugates harboring a kinesin spindle protein inhibitor with immunostimulatory properties

Figures & data

Figure 1. Scheme of TWEAKR-KSPi-ADCs, its metabolite and the small molecule KSPi.

The tumor necrosis factor-like weak inducer of apoptosis (TWEAKR) targeting, agonistic antibody BAY-356, which is cross-reactive to human and murine TWEAKR, is conjugated via a protease-cleavable linker with a small molecule inhibitor of kinesin family member 11 (KIF11, kinesin-5, BimC, Eg5, best known as KSP). The resulting TWEAKR-KSPi-ADCs are depicted as TWEAKR-KSPi-ADC1 (a) linked via lysine residues to the antibody and TWEAKR-KSPi-ADC2 (b) linked via cysteine residues to the antibody. Thus, TWEAKR-KSPi-ADC1 and TWEAKR-KSPi-ADC2 differ solely in how the linker-payload is attached to the antibody BAY-356. Upon legumain-mediated cleavage in the lysosomal compartment, the active metabolite (c) is formed, which shows poor cellular permeability. The cell-permeable small molecule KSPi (d) was used for investigation of ICD in in vitro studies.

Figure 2. TWEAKR-KSPi-ADC controls tumor growth in an immune-dependent fashion in TWEAKR expressing CT26.

(a,b) Murine colon cancer CT26 cells were inoculated into Balb/c mice. Palpable subcutaneous tumors were resected and immunohistochemistry employing an anti-TWEAKR antibody was conducted. Size bars indicate 2 mm (top), 500 μm (middle) and 100 μm (bottom). (c) Murine colon cancer CT26 cells were inoculated into Balb/c (d) or NOD-SCID (e) mice. Mice bearing palpable subcutaneous tumors were treated with a single dose of either 2.5 mg/kg of TWEAKR-KSPi-ADC1 or 5 mg/kg of the anti-CTLA-4 antibody via i.p. injection. The vehicle PBS was applied with a volume of 5 ml/kg. TWEAKR-KSPi-ADC1 showed a significant (p < .0001) tumor growth inhibition comparable to the effect of the anti-CTLA-4 antibody in the immunocompetent mice, yet had no effect in immunodeficient NOD-SCID mice. Statistics were performed by One-Way ANOVA using log-transformed final tumor volumes followed by a Dunnett’s multiple comparisons test. P-values equal * p < 0,05; ** p < 0,01; *** p < 0,001; n = 8–10).

Table 1. In vivo treatment schedule and outcome

Tumor model; mouse strain	TWEAKR-KSPi-ADC	Dose (mg/kg)	Schedule	T/Cvol (final)	Note
CT26; Balb/c	ADC1	2.5	Single dose	0.25	Figure 2d
CT26; NOD/SCID	ADC1	2.5	Single dose	0.89	Figure 2e
CT26; Balb/c	ADC2	5	Biweekly	0.08	Figure 5b
MC38; C57Bl/6	ADC2	5	Biweekly	0.31	Fig. S1D
MC38; NOD/SCID	ADC2	5	Biweekly	0.38	Fig. S1E

Murine colon carcinoma CT26 and MC38 cells were inoculated in the flank of syngeneic Balb/c and C57Bl/6 mice, respectively. Alternatively, CT26 or MC38 cells were inoculated in immunodeficient NOD/SCID mice. When subcutaneous tumors became palpable animals were treated via intraperitoneal (i.p.) injections either with a single dose of 2.5 mg/kg TWEAKR-KSPi-ADC1 or with biweekly 5 mg/kg TWEAKR-KSPi-ADC2 for two weeks, respectively. The tumor area was measured by calipers three times per week followed by calculation of the tumor volume (T_vol = 0.5 x length x width²). T/C_vol served as a parameter describing the ratio of mean tumor volumes of treated mice compared to the volume of vehicle control treated mice.

Figure 3. Prediction of the potential of KSPi to induce immunogenic cell death.

The immunogenic cell death (ICD) scores were predicted for each compound from the NCI60 collection together with the KSPi used in this study by means of the ICDPred package (https://github.com/kroemerlab/ICDpred).^19,51 The distribution of ICD scores was plotted (dashed lines indicate quantiles) showing that the KSPi and the prototype ICD inducer mitoxantrone (MTX) belong to the 20% and 10% highest scores, respectively.

Figure 4. Biosensor cells for the immunogenic cell death fingerprinting (a-c) Human colorectal adenocarcinomas HT29 cells were either stained with quinacrine to assess ATP release, or were genetically modified to express CALR-GFP as surrogate marker for CALR exposure, HMGB1-GFP to measure nuclear HMGB1 exodus or GFP expression under the control of the type-I interferon-induced GTP-binding protein MX1 promoter to measure type-I interferon responses in a high throughput fashion. A schematic representation is depicted in (a) and representative images of HT29 cells untreated (CTRL) or treated with the KSPi or mitoxantrone are shown in (b). Size bar equals 10 µm. (c) To assess ICD fingerprints, HT29 cells were treated with the indicated concentrations of KSPi. Mitoxantrone and recombinant IFNα at the indicated concentrations were used as positive controls. The data is plotted together with solvent controls (vehicle) in form of a heatmap. White boxes indicate: “data not available”.

Hallmarks of ICD were measured by means of biosensors on a fluorescence-based phenotypic screening platform as described above. Altogether the KSPi was as efficient as MTX in inducing the four measured ICD hallmarks in a dose- and time-dependent fashion.

Figure 5. TWEAKR-KSPi-ADC triggers anticancer immune responses.

Murine colon carcinoma CT26 cells were inoculated in the flank of syngeneic Balb/c mice. When subcutaneous tumors were palpable animals were treated twice per week with either 5 mg/kg TWEAKR-KSPi-ADC2 or corresponding isotype control KSPi-ADC, or 5 mg/kg anti-CTLA-4 via intraperitoneal (i.p.) injections for two weeks. (a) TWEAKR-KSP-ADC2 showed a significant (p < .0001) tumor growth inhibition with a final T/C_vol of 0.08, even stronger than anti-CTLA-4 mAb (T/C_vol 0.28; p < .0001). Isotype- control KSPi-ADC had no significant effect on tumor growth (b). Statistics were performed by One-Way ANOVA using log-transformed final tumor volumes followed by a Dunnett’s multiple comparisons test. P-values equal * p < 0,05; ** p < 0,01; *** p < 0,001 (n = 8–10). Ex vivo flow cytometry of CT26 tumors treated with TWEAKR-KSPi-ADC2: Ex vivo analysis of CT26 tumor samples via flow cytometry demonstrated an increase of CD45⁺ leukocyte frequency (c) in TWEAKR-KSPi-ADC2 treated mice as well as an increase of CD4⁺ and CD8⁺ T cell frequency, which was increased compared to treatment with anti-CTLA-4 (d,e). Moreover, the frequency of immunosuppressive CD4⁺CD25⁺FoxP3⁺ Treg cells was reduced by TWEAKR-KSPi-ADC2 (f). In line with the flow cytometry results, the proinflammatory cytokines IFNγ, IL-2, and TNFα were found to be enhanced in tumor samples of TWEAKR-KSPi-ADC2 treated mice on the last day of the experiment (g–i).

Table

Antibody	Fluorochrome	Company	REF No.	Dilution
Set #1
CD45	APC-Cy7	BD Pharmingen	557659	1:200
CD4	eFluor450	eBioscience	48-0042-82	1:100
CD8a	APC	BD Pharmingen	553035	1:100
CD25	Alexa Fluor 488	eBioscience	53-0251-82	1:100
NKp46	PE-Cy7	BioLegend	137618	1:200
Ki-67	PerCP-eFluor710	eBioscience	46-5698-82	1:200
FoxP3	PE	eBioscience	12-5773-82	1:100
Set #2
CD45	APC-Cy7	BD Pharmingen	557659	1:200
CD11b	eF450	eBioscience	48-0112-82	1:300
GR 1	APC	Miltenyi	130-102-838	1:300
MHC II	PerCP	BioLegend	107624	1:200
CD11c	PE-Cy7	BioLegend	117318	1:200
F4/80	PE	eBioscience	12-4801-82	1:200
CD206	FITC	BioLegend	141704	1:200

Table

ADC	antibody–drug conjugate
ATP	adenosine triphosphate
CALR	calreticulin
ER	endoplasmic reticulum
GFP	green fluorescent protein
ICD	immunogenic cell death
IFN	interferon
KSPi	kinesin spindle protein inhibitor
MTX	mitoxantrone

Kepp O, Sauvat A, Leduc M, Forveille S, Liu P, Zhao L, Bezu L, Xie W, Zitvogel L, and Kroemer G, . A fluorescent biosensor-based platform for the discovery of immunogenic cancer cell death inducers. Oncoimmunology. 2019;8(8):1606665. doi:10.1080/2162402X.2019.1606665.

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