The development of antibody drug conjugates (ADCs) for the
treatment of acute myeloid leukemia (AML) has been limited by the availability
of unique disease-specific antigens. Gemtuzumab ozogamicin is the only ADC
approved for use in CD33
+ AML patients,
but is associated with prolonged cytopenias, presumably because of its effects
on CD33
+ hematopoietic
progenitor cells. The use of bispecific antibodies represents a new approach to
the development of more highly tumor-targeted ADCs. Phenotyping of AML patient
samples indicates that CD33, a myeloid cell marker, and CD7, a lymphoid cell
marker, are co-expressed in approximately 25% of AML patients.
Methods
Fabs directed against CD7 and
CD33, each with a specific mutation to introduce a cysteine residue at codon
207, were expressed in HEK293 cells, and affinities assessed using SPR. Using
BiVictriX’s BiCygni®technology platform, anti-CD7,
anti-CD33 Bi-Fabs were produced through conjugation of DBCO and azide regents
to the CD7 and CD33 Fabs respectively, and cross-linked using click chemistry.
Bi-Fabs were conjugated to vcMMAE and mcMMAF through cysteine reduction and
conjugation. Bi-Fab drug conjugates were assessed for cytotoxicity against CD7+CD33+, CD7+CD33-, CD7-CD33+, and CD7-CD33- cells
and in colony forming assays, with corresponding homo-dimeric anti-CD7 and
anti-CD33 bi-Fab and gemtuzumab drug conjugates with equivalent DARs used as
controls.
Results
The wild-type Fabs used as starting points were shown to have Kd values
of 3.6nM (CD7 Fab) and 1.6 nM (CD33 Fab) for their respective protein targets.
The resulting anti-CD7, anti-CD33 Bi-Fab MMAE conjugate (BVX100-MMAE) was shown
to be highly cytotoxic to HNT-34 and Kasumi-3 (CD7+CD33+)
cells with CC50 values of 0.20±0.04nM and 0.20±0.03nM respectively, compared to CC50 values of 1.3nM and
1.2nM for gemtuzumab MMAE. BVX100-MMAE showed 5-fold lower cytotoxicity towards
Jurkat cells (CD7+CD33-), >10-fold margins relative to
MV4.11 and SHI-1 (CD7-CD33+) cells, and had no measurable
cytotoxicity against DND-39 (CD7-CD33-) cells at
concentrations up to 10nM. In contrast, the corresponding homodimeric
bi-Fab-MMAE conjugate showed similar levels of activity against HNT-34 and
Kasumi-3 cells as BVX100-MMAE, but no selectivity relative to cells expressing
only CD7 (anti-CD7 Bi-Fab) or CD33 (anti-CD33 bi-Fab). BVX100-MMAF showed no
inhibition of colony formation in a GM-CFU assay at concentrations up to 10nM,
whereas gemtuzumab-MMAF showed 60% and >90% reductions in colony formation
at concentrations of 1nM and 10nM respectively. Targeted mutagenesis within the
CDRs of the anti-CD7 Fab to reduce affinity for CD7, and subsequent conjugation
of these mutant Fabs to the wild-type anti-CD33 Fab, led to the identification
of BVX-130-MMAE and -MMAF, which retain sub-nM potency against HNT-34 and
Kasumi-3 cells, but have substantially higher selectivity for these CD7+CD33+ cells
compared to Jurkat, MV4,11 and SHI-1 cells.
Conclusion
Anti-CD7, anti-CD33 Bi-Fab drug conjugates show high potency against CD7+CD33+ cells,
and have substantially greater selectivity than monospecific drug conjugates
for such dual positive cells. The in vivo efficacy of BVX130-MMAF is presently
being assessed in a xenograft model using HNT-34 cells. In parallel with the
advancement of the lead CD7/CD33 project, BiVictriX’s BiCygni® technology platform is currently being applied to the initia
