The chimeric monoclonal antibody cAC10, directed against CD30, induces growth arrest of CD30 ؉ cell lines in vitro and has pronounced antitumor activity in severe combined immunodeficiency (SCID) mouse xenograft models of Hodgkin disease. We have significantly enhanced these activities by conjugating to cAC10 the cytotoxic agent monomethyl auristatin E (MMAE) to create the antibody-drug conjugate cAC10-vcMMAE. MMAE, a derivative of the cytotoxic tubulin modifier auristatin E, was covalently coupled to cAC10 through a valine-citrulline peptide linker. The drug was stably attached to the antibody, showing only a 2% release of MMAE following 10-day incubation in human plasma, but it was readily cleaved by lysosomal proteases after receptormediated internalization. Release of MMAE into the cytosol induced G 2 /Mphase growth arrest and cell death through the induction of apoptosis. In vitro, cAC10-vcMMAE was highly potent and selective against CD30 ؉ tumor lines (IC 50 less than 10 ng/mL) but was more than 300-fold less active on antigennegative cells. In SCID mouse xenograft models of anaplastic large cell lymphoma or Hodgkin disease, cAC10-vcMMAE was efficacious at doses as low as 1 mg/kg. Mice treated at 30 mg/kg cAC10-vcMMAE showed no signs of toxicity. These data indicate that cAC10-vcMMAE may be a highly effective and selective therapy for the treatment of CD30 ؉ neoplasias.

The B-lymphocyte-restricted glycoprotein CD22 is expressed on mature IgM+IgD+ B cells, and is capable of binding to ligands on T and B cells. CD22 can interact with both the B-cell antigen receptor (BCR) complex and signalling molecules, including the protein tyrosine phosphatase SHP1 (PTP1C, SHP), a putative negative regulator of BCR signalling. Thus CD22 may facilitate interactions with lymphocytes and regulate the threshold of BCR signalling. To define the in vivo function of CD22, we generated CD22-deficient mice. Here we show that CD22 is required for normal antibody responses to thymus-independent antigens and regulates the lifespan of mature B cells.

A highly cytotoxic DNA cross-linking pyrrolobenzodiazepine (PBD) dimer with a valine-alanine dipeptide linker was conjugated to the anti-CD70 h1F6 mAb either through endogenous interchain cysteines or, site-specifically, through engineered cysteines at position 239 of the heavy chains. The h1F6239C-PBD conjugation strategy proved to be superior to interchain cysteine conjugation, affording an antibody-drug conjugate (ADC) with high uniformity in drug-loading and low levels of aggregation. In vitro cytotoxicity experiments demonstrated that the h1F6239C-PBD was potent and immunologically specific on CD70-positive renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines. The conjugate was resistant to drug loss in plasma and in circulation, and had a pharmacokinetic profile closely matching that of the parental h1F6239C antibody capped with N-ethylmaleimide (NEM). Evaluation in CD70-positive RCC and NHL mouse xenograft models showed pronounced antitumor activities at single or weekly doses as low as 0.1 mg/kg of ADC. The ADC was tolerated at 2.5 mg/kg. These results demonstrate that PBDs can be effectively used for antibody-targeted therapy.

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinasedependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase C ␥ 2 are early events in collageninduced activation. We recently proposed that collagensignaling in platelets involves a receptor or a receptorassociated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor ␥-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor ␥-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor ␥-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin ␣ 2 ␤ 1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, Fc␥RIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor ␥-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin ␣ 2 ␤ 1 and involves phosphorylation of the Fc receptor ␥-chain, its association with Syk and subsequent phosphorylation of phospholipase C ␥ 2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.The adhesive and stimulatory properties of the extracellular matrix protein collagen on platelets are vital for the maintenance of hemeostasis. Upon vascular damage, platelets adhere to subendothelial collagen which stimulates a tyrosine kinase dependent pathway leading to platelet degranulation, aggregation and development of a hemeostatic plug. The mechanism of collagen stimulation of platelets is poorly understood, and the distinction between adhesion and stimulation ill defined. Several platelet glycoproteins have been implicated as potential collagen receptors, including the integrin ␣ 2 ␤ 1 (1), glycoprotein IV (GPIIIb, CD36) (2), glycoprotein VI (3), and uncharacterized 65-kDa (4) and 85-90-kDa glycoproteins (5). Patients whose platelets express abnormally low numbers of these proteins, or who possess autoantibodies to them, have limited bleeding defects (3, 5-9).Collagen stimulation of platelets activates tyrosine kinasedependent mechanisms which involve tyrosine phosphorylation of Syk and phospholipase C ␥ 2 (PLC ␥ 2) 1 (10 -12). Syk is a nonreceptor tyrosine kinase which is assembled into signaling complexes via interaction between its tandem Src homology 2 (SH2) domains and a tyrosine phosphorylated activation motif found in receptors of the immune system or their associated chains. The motif, termed the immunoreceptor tyrosine-based activation motif (ITAM), has the ...