Horizontal Transmission of Cytomegalovirus in a Rhesus Model Despite High-Level, Vaccine-Elicited Neutralizing Antibody and T-Cell Responses.

The development of a vaccine to prevent congenital human cytomegalovirus (HCMV) disease is a public health priority. We tested rhesus CMV (RhCMV) prototypes of HCMV vaccine candidates in a seronegative macaque oral challenge model. Immunogens included a recombinant pentameric complex (PC; gH/gL/pUL128/pUL130/pUL131A), a postfusion gB ectodomain, and a DNA plasmid that encodes pp65-2. Immunization with QS21-adjuvanted PC alone or with the other immunogens elicited neutralizing titers comparable to those elicited by RhCMV infection. Similarly, immunization with all 3 immunogens elicited pp65-specific cytotoxic T-cell responses comparable to those elicited by RhCMV infection. RhCMV readily infected immunized animals and was detected in saliva, blood, and urine after challenge in quantities similar to those in placebo-immunized animals. If HCMV evades vaccine-elicited immunity in humans as RhCMV evaded immunity in macaques, a HCMV vaccine must elicit immunity superior to, or different from, that elicited by the prototype RhCMV vaccine to block horizontal transmission.

Determination of pharmacokinetic values of calicheamicin-antibody conjugates in mice by plasmon resonance analysis of small (5 microl) blood samples.

PURPOSE: The present study aims to establish a method that provides fast, precise and reproducible pharmacokinetic (PK) parameters of antibody-calicheamicin conjugates. The method should discriminate between PK of the antibody moiety and PK of the conjugated calicheamicin (CM). METHODS: The conjugates gemtuzumab ozogamicin (CMA-676, Mylotarg) or inotuzumab ozogamicin (CMC-544) were injected in the tail vein of nude mice. At regular time intervals, 5 mul whole blood samples were taken from the tail artery. Concentrations of conjugated CMA-676 or CMC-544 as well as concentrations of their respective antibody moiety were determined by sandwich plasmon resonance. This detection system measures changes in the plasma resonance angle caused by the interaction of macromolecules on biosensor chips. We determined as a first measure the binding of CMA-676 or CMC-544 to their respective antigens, CD33 or CD22. As a second measure we determined the amount of CM on the antigen-bound conjugates. This was done by determination of changes in plasma resonance angle after binding of an anti-CM antibody. RESULTS: Sandwich plasmon resonance allowed detection of both conjugates in blood of mice in a range of 100-1,000 ng/ml protein. Due to the precision of the sampling and detection methods, PK values of each conjugate were determined in individual mice. Calicheamicin bound to antibody was eliminated faster than the antibody alone. The presence of a CD22-expressing tumour in mice reduced the plasma levels of the CD22-targeting conjugate but not of the CD33-targeting one. CONCLUSIONS: Using small blood samples from a mouse, the sandwich plasmon resonance method provided PK-values of CM-conjugates and information about the stability of the linkage in vivo. Comparison between the PK-values of CM-conjugates in tumour-bearing and tumour-free mice suggested that retention of the conjugate in tumour tissue due to antigen targeting could be deduced from the plasma levels.

Antitumor efficacy of a combination of CMC-544 (inotuzumab ozogamicin), a CD22-targeted cytotoxic immunoconjugate of calicheamicin, and rituximab against non-Hodgkin's B-cell lymphoma.

PURPOSE: CMC-544 is a CD22-targeted cytotoxic immunoconjugate, currently being evaluated in B-cell non-Hodgkin's lymphoma (B-NHL) patients. Rituximab is a CD20-targeted antibody commonly used in B-NHL therapy. Here, we describe antitumor efficacy of a combination of CMC-544 and rituximab against B-cell lymphoma (BCL) in preclinical models. EXPERIMENTAL DESIGN: BCLs were cultured in vitro with CMC-544, rituximab, or their combination. BCLs were injected either s.c. or i.v. to establish localized s.c. BCL in nude mice or disseminated BCL in severe combined immunodeficient mice, respectively. I.p. treatment with CMC-544 or rituximab was initiated at various times either alone or in combination and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. RESULTS: In vitro growth-inhibitory activity of CMC-544 combined with rituximab was additive. Rituximab but not CMC-544 exhibited effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Rituximab was less effective in inhibiting growth of established BCL xenografts than developing xenografts. In contrast, CMC-544 was equally effective against both developing and established BCL xenografts. Although CMC-544 and rituximab individually caused partial inhibition of the growth of BCL xenografts at suboptimal doses examined, their combination suppressed xenograft growth by >90%. In a disseminated BCL model, 60% of CMC-544-treated mice and 20% of rituximab-treated mice survived for 125 days. In contrast, 90% of mice treated with the combination of CMC-544 and rituximab survived for longer than 125 days. CONCLUSION: The demonstration of superior antitumor activity of a combination of CMC-544 and rituximab described here provides the preclinical basis for its clinical evaluation as a treatment option for B-NHL.

Antibody-targeted chemotherapy with CMC-544: a CD22-targeted immunoconjugate of calicheamicin for the treatment of B-lymphoid malignancies.

Antibody-targeted chemotherapy with gemtuzumab ozogamicin (CMA-676, a CD33-targeted immunoconjugate of N-acetyl-gamma-calicheamicin dimethyl hydrazide [CalichDMH], a potent DNA-binding cytotoxic antitumor antibiotic) is a clinically validated therapeutic option for patients with acute myeloid leukemia (AML). Here, we describe the preclinical profile of another immunoconjugate of CalichDMH, CMC-544, targeted to CD22 expressed by B-lymphoid malignancies. CMC-544 comprises a humanized IgG4 anti-CD22 monoclonal antibody (mAb), G5/44, covalently linked to CalichDMH via an acid-labile 4-(4'-acetylphenoxy) butanoic acid (AcBut) linker. Both CMC-544 and unconjugated G5/44 bound human CD22 with subnanomolar affinity. CMC-544, but not unconjugated G5/44, exerted potent cytotoxicity against CD22+ B-cell lymphoma (BCL) cell lines (inhibitory concentration of 50%: 6-600 pM CalichDMH). CMC-544 caused a potent inhibition of growth of small but established BCL xenografts leading to cures (therapeutic index > 10). CMC-544 prevented the establishment of BCL xenografts and also caused regression of large BCLs (> 1.5 g tumor mass). In contrast, unconjugated CalichDMH, unconjugated G5/44, and an isotype-matched control conjugate, CMA-676, were ineffective against these BCL xenografts. Thus, CD22-targeted delivery of CalichDMH is a potent and effective preclinical therapeutic strategy for BCLs. The strong antitumor profile of CMC-544 supports its clinical evaluation as a treatment option for B-lymphoid malignancies.