Melanoma cells can be eliminated by sialylated CD43 × CD3 bispecific T cell engager formats in vitro and in vivo

Abstract

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3ε. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.

Keywords: Antibody immunotherapy; Bispecific T-cell engaging antibodies; Breast cancer; Hematological cancers/leukemias; Post-translationally modified drug target; Skin cancers/melanoma.

Fig. 1

AT1413 binds to melanoma and breast cancer cell lines and patient-derived melanoma samples. a Representative normalized histograms for melanoma, leukemia, breast cancer, colon carcinoma and pancreas carcinoma cell lines. Black lines indicate 1.25 µg/ml AT1413 and grey curves the negative control antibody AT1002. b Delta MFI = MFI AT1413-AT1002 on short term cultured patient-derived melanoma samples with the BLM cell line included as a reference. Antibody concentration of 1 µg/ml. Shading indicates samples also used in ADCC in Fig. 3

Fig. 2

Immunohistochemistry staining with AT1413-biotin at 10 µg/ml on formalin-fixed paraffin-embedded melanoma microarray. AT1413 staining in green, counterstaining with hematoxylin. a stage III tumor on the upper arm; b stage II tumor on the sole of the foot; c Stage III tumor of the scalp; d Adjacent healthy skin of the foot

Fig. 3

Immunoprecipitation confirms that AT1413 recognizes CD43 on melanoma cell lines. AT1413 immunoprecipitates CD43 from a melanoma 136.2 cells as detected by mouse anti-CD43 (clone MEM59; top panel) and with rabbit anti-CD43 (clone SP55; bottom panel). b IP of A375 as detected by mouse anti-CD43 (clone MEM59). c Removing the sialic acids by neuraminidase treatment of the cells results in diminished binding of AT1413 to these cells. Black lines indicate 1.0 µg/ml AT1413 and grey curves the negative control antibody AT1002. IP: immunoprecipitate

Fig. 4

Cytotoxicity was tested at different AT1413 concentrations and for the unmodified antibody on indicated cell lines. a ADCC in DELFIA^® cell cytotoxicity assay (PerkinElmer; E:T ratio 50:1; correspondening to 3 NK cells per target cell) at an indicated concentration or b in chromium-51 assays at 25 µg/ml on primary melanoma cultures (E:T ratio 33:1 of T-cell depleted PBMC; correspondening to around 10 NK cells per target cell). ADCC: antibody-dependent cellular cytotoxicity, E:T ratio: effector-to-target cell ratio

Fig. 5

T-cell mediated cytotoxicity by different T-cell engaging formats of AT1413. a Cytototoxicity by the bTCE format on two different melanoma cell lines (E:T ratio 10:1). b Schematic representation of the double bivalent (2 + 2) bispecific bTCE-format (left) and the double monovalent (1 + 1) bispecific KiH-format (left). c T-cell mediated cytotoxicity of A375 cells by AT1413 bTCE compared to AT1413 KiH. d CD25 and CD69 activation marker expression on T cells incubated with AT1413 bTCE, AT1002 bTCE, AT1413 KiH or AT1002 KiH in the presence of A375 target cells. e Time-course of T-cell mediated toxicity induced by AT1413 bTCE or KiH (E:T ratio 10:1). Error bars represent duplicate or triplicate measurements within the experiment. bTCE: bivalent T cell engaging antibody, E:T ratio: effector-to-target cell ratio, KiH: knob-in-hole

Fig. 6

Specific tumor growth inhibition and T-cell activation in vivo. a Normalized tumor growth (fold increase of original tumor at start of treatment) of A375 melanoma cell line engrafted on both flanks in HIS mice. Black arrow indicates the start of treatment when the mean tumor volume was 39.3 ± 25.5 mm³. Mice received either 50 μg AT1413 bTCE or control, or 31 μg AT1413 KiH or control. Growth difference between AT1413 bTCE and control P = 0.0056; and between AT1413 KiH and control P = 0.0405 (two-way ANOVA/mixed-effect analysis). b Percentage of T cells within total live cells in peripheral blood, bone marrow, spleen or tumor at a sacrifice. c Percentage lymphocytes with an activated phenotype as detected by CD38⁺CD69⁺ in the same tissues as for (b) at a sacrifice. Error bars represent mean with a standard deviation of the mice per treatment group. *P < 0.05, **P < 0.01. Statistically significant were indicated as evaluated by two-way ANOVA/mixed-effect analysis, corrected for multiple testing (AT1413 bTCE compared to control bTCE, and AT1413 KiH compared to KiH control). S.C. subcutaneous, I.V. intravenous, HIS Humanized immune system, BM bone marrow, PB peripheral blood, SPL spleen, TUM tumor