Preclinical Development of a Novel Humanized and Fc-Engineered Antibody-Drug Conjugate for the Therapy of T-Cell Acute Lymphoblastic Leukemia
Authors: Krohn Steffen , Gehlert Carina Lynn , Winterberg Dorothee , Jochimsen Alexander , Boje Ammelie Svea , Baum Natalie , Baldus Claudia D , Brüggemann Monika , Cario Gunnar , Schrappe Martin , Lenk Lennart , Gramatzki Martin , Klausz Katja , Kellner Christian , Peipp Matthias 
Published: December 2024

In the last decades the outcome for patients with T-cell acute lymphoblastic leukemia (T-ALL) has improved significantly, but therapeutic options for high-risk subtypes and poor responders to standard therapy are still limited. Especially approved antibody-based or cellular immunotherapies are lacking. CD7 represents a promising target structure that is expressed in all T-ALL subtypes including early T-cell precursor (ETP)-ALL. Different approaches targeting CD7 in T-ALL are currently under development, including CAR-T cell therapy. Since CD7 shows a high internalization capacity, it represents a very promising target structure for antibody-drug conjugates (ADC). Recently, we demonstrated that the Fc-optimized ADC, chimTH69-DE-vcMMAE, which is based on a chimeric CD7 antibody, has significant antileukemic activity in vitro and in vivo due to its unique set of Fc-mediated and payload-mediated effector functions.

In the current study, the antibody backbone was humanized to facilitate further clinical development. A combination of complementarity determining region (CDR)-grafting and phage display-based guided selection of a fully human light chain was applied. The final humanized antibody, huTH69, showed outstanding biological characteristics and a high degree of developability in terms of level of humanness (T20 score (VH/VL) = 88.6/91.1; OASis identity (VH+VL) = 87%), thermal stability and resistance to post-translational modifications (e.g., aspartic acid isomerization). HuTH69 revealed high affinity binding to recombinant CD7 (KD = 1.8 nM) and dose-dependent binding to CD7-positive ALL cells similar to the parental antibody. Interestingly, the humanized antibody more efficiently triggered complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) compared to a panel of published CD7 antibodies. ADCC activity was further increased by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc part (EC50 ADCC, 2.62-12.75 pM). The capacity to trigger Fc-mediated effector functions more efficiently than a panel of other published CD7 antibodies could probably be attributed to a unique epitope on CD7 that is bound by huTH69, which we defined on the Ig-like domain by site-directed mutagenesis mapping.

The humanized and Fc-engineered antibody huTH69-DE was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) using an enzymatic-cleavable linker (mc-vc-PABC). The novel ADC huTH69-DE-vcMMAE, which displayed a drug-to-antibody ratio of 3.2, showed strong growth inhibitory effects at subnanomolar concentrations and high efficiency against different T-ALL cell lines with varying CD7 antigen densities (IC50 0.13-1.79 nM). Interestingly, the humanized ADC was significantly more active than the parental ADC on leukemia cells expressing lower levels of CD7. Growth inhibition was due to G2 cell cycle arrest and induction of apoptosis. The in vivo activity of huTH69-DE-vcMMAE was investigated in a xenograft mouse model using CEM T-ALL cells that were subcutaneously injected into immunocompromised NSG mice. In comparison to untreated animals and mice treated with a similarly designed control ADC (IgG1-DE-vcMMAE), huTH69-DE-vcMMAE led to significantly reduced tumor growth resulting in significantly prolonged overall survival. No obvious signs of unspecific toxicity (e.g., loss of body weight) were observed.

Taken together, the novel CD7-targeting ADC huTH69-DE-vcMMAE showed an outstanding high degree of developability, binds a unique CD7 epitope, triggers a unique set of effector functions and demonstrated potent antitumor activity in vivo. The described ADC is a promising leading candidate for entering further clinical development.