Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22

Abstract

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22+ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.

Fig. 1a–c

Modulation of mAbs bound to CD22 expressed on the surface of normal peripheral blood–derived B cells and BCLs. Anti-CD22 mAbs were allowed to bind at 4°C to either Daudi BCLs (a) or normal peripheral blood B cells (b), and washed to remove unbound mAb. The B cells were further maintained at 37°C for varying lengths of time up to 30 min after which the surface retention of the anti-CD22 mAb was assessed by indirect immunofluorescence analysis. A similar evaluation was conducted using three distinct BCLs (c), except that the modulation period was extended to 24 h

Fig. 2

Structural representation of the linkers used to conjugate CalichDM derivatives to the targeting mAb. Both the AcBut- and AcPAc-linked conjugates include an acid-labile hydrazone functionality. The amide-linked conjugate lacks such an acid-labile group

Fig. 3A–C

Effect of CalichDM conjugated to m5/44 on the growth of Ramos BCL xenografts. Subcutaneous tumors were established prior to the administration of CalichDM conjugates at a fixed dose of 160 μg of conjugated CalichDM/kg. Three such doses were administered 4 days apart (Q4D×3). CD33-targeted conjugate of CalichDMH (CMA-676) was used as a nonspecific conjugate. Unconjugated m5/44 at 8 mg/kg was used as a control. Three separate studies are presented in panels A, B, and C. Results are presented as mean tumor mass ± SEM

Fig. 4

Effect of CalichDM conjugated to m5/44 on the growth of Raji BCL xenografts. Subcutaneous tumors were established to an average mass of 300 mg after which CalichDM conjugates of m5/44 were administered at 160 μg of conjugated CalichDM/kg. Three such doses were administered 4 days apart (Q4D×3). The CD33-targeted conjugate of CalichDMH (CMA-676) was used as a nonspecific conjugate. Unconjugated m5/44 at 8 mg/kg was used as a control. Results are presented as mean tumor mass ± SEM

Fig. 5a,b

The sequences of the m5/44 variable heavy region (VH, VL) are aligned with the human germline sequence (DP-7, DPK-9). Asterisks indicate differences between mouse (donor) and human (acceptor) residues. CDRs are indicated in blue (not shown for acceptor frameworks), mouse framework residues are colored red, and human residues are shown in black. The grafted gH and gL sequences are shown below the human germline sequence. Sequences underlined and colored red indicate donor residues that have been retained in the graft. The underlined residues in CDR-H2 show potential sites for glycosylation and calicheamcin conjugation. Residue numbering is according to Kabat [31]. The variable domain sequences of the light and heavy chains of the murine antibody m5/44 and its humanized counterparts have been deposited in the Genbank with the following accession number: m5/44 kappa light chain VJ region, AY531629 (protein ID number AAS45201), g5/44 kappa light chain VJ region gLa, AY531630 (protein ID number AAS45202), g5/44 kappa light chain VJ region gLb, AY531631 (protein ID number AAS45203), m5/44 heavy chain VDJ region, AY531632 (protein ID AAS45204), g5/44 heavy chain VDJ region gHa, AY531633 (protein ID number AAS45205), g5/44 heavy chain VDJ region gHb, AY531634 (protein ID number AAS45206), g5/44 heavy chain VDJ region gHc, AY531635 (protein ID number AAS45207), g5/44 heavy chain VDJ region gHd, AY531636 (protein ID number AAS45208)

Fig. 6a,b

Competitive inhibition of the binding of murine anti-CD22 mAb, m5/44, to Ramos BCL by its humanized versions assembled by combinations of four distinct grafts of heavy chain (gHa–d) and two distinct grafts of light chain (gLa, gLb). Increasing concentrations of the humanized mAb were mixed with the fixed concentration of m5/44 and then allowed to bind to Ramos BCL. Binding of the murine mAb, m5/44, was monitored by indirect immunofluorescence analysis using flow cytometry. The chimeric version of m5/44 (cL cH) was also used in this evaluation as a positive control

Fig. 7

Effect of humanized anti-CD22 mAb g5/44 on the binding of various murine anti-CD22 mAbs to human CD22-mFc detected by biosensor analysis. Various murine anti-CD22 mAbs were individually (50 μg/ml) allowed to bind CD22-mFc immobilized on biosensor chip that had been pretreated (100 μg/ml) with either anti-CD22 mAb g5/44 or anti-CD33 mAb hP67.6 (control mAb). Percentage Inhibition of murine mAb binding was calculated as 100×[1−(RU with g5/44 pretreatment) / (RU with hP67.6 pretreatment)]

Fig. 8

Effect of calicheamicin conjugates of murine and humanized anti-CD22 mAbs. Subcutaneous BCLs were established prior to the i.p. administration of CalichDM conjugates at either 10 or 160 μg of conjugated CalichDM/kg. Three such doses were administered 4 days apart (Q4D×3). Unconjugated humanized anti-CD22 mAb, g5/44, was administered i.p. at 8 mg/kg Q4D×3 as a control. Results from a representative experiment are presented as mean tumor mass (g) ± SEM. The number of tumor-free mice is indicated wherever appropriate