Plasma and cerebrospinal fluid pharmacokinetics of vodobatinib, a neuroprotective c-Abl tyrosine kinase inhibitor for the treatment of Parkinson's disease.

BACKGROUND: Preclinical evidence suggests that c-Abl is critical in the pathogenesis of Parkinson's Disease (PD). Vodobatinib (K0706) is a potent, specific Abl kinase inhibitor currently being developed for the treatment of PD. In previously reported studies, nilotinib, a multikinase c-Abl inhibitor, did not show clinical activity as evidenced by no improvement of symptoms or the rate of decline after one to six months of treatment at the maximum permissible dose, presumably because of insufficient CNS penetration. Here we report clinical PK and safety data for vodobatinib. OBJECTIVES: To determine safety, plasma PK, and CSF penetration of vodobatinib in healthy volunteers and PD subjects following oral administration, and compare CSF levels to in vitro concentrations required for c-Abl inhibition relative to data reported for nilotinib. METHODS: Inhibition of c-Abl kinase activity and c-Abl binding affinity were first assessed in vitro. Healthy human volunteers and PD patients received various oral doses of vodobatinib once-daily for seven and fourteen days respectively, to assess safety, and plasma and CSF PK. RESULTS: In in vitro assays, vodobatinib was more potent (kinase IC50 = 0.9 nM) than nilotinib (kinase IC50 = 15-45 nM). Administration of vodobatinib 48, 192 and 384 mg to healthy subjects for 7 days yielded mean Cmax, CSF values of 1.8, 11.6, and 12.2 nM respectively, with the two highest doses exceeding the IC50 over the entire dosing interval. Cavg, CSF values were 6-8 times greater than the IC50. Comparable CSF levels were observed in PD patients. All doses were well tolerated in both cohorts. CONCLUSION: Based on achieved CSF concentrations, the potential for c-Abl inhibition in the brain is substantially higher with vodobatinib than with nilotinib. The CSF PK profile of vodobatinib is suitable for determining if c-Abl inhibition will be neuroprotective in PD patients.

ASN004, A 5T4-targeting scFv-Fc Antibody-Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models.

The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors but shows very limited expression in normal adult tissues. ASN004 is a 5T4-targeted antibody-drug conjugate (ADC) that incorporates a novel single-chain Fv-Fc antibody and Dolaflexin drug-linker technology, with an Auristatin F hydroxypropylamide payload drug-to-antibody ratio of approximately 10-12. The pharmacology, toxicology, and pharmacokinetic properties of ASN004 and its components were investigated in vitro and in vivo ASN004 showed high affinity for the 5T4 antigen and was selectively bound to and internalized into 5T4-expressing tumor cells, and potent cytotoxicity was demonstrated for a diverse panel of solid tumor cell lines. ASN004 induced complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a wide range of 5T4 expression levels. A single dose of ASN004, as low as 1 mg/kg i.v., achieved complete tumor regression leading to tumor-free survivors in the A431 cervical cancer model. In head-to-head studies, superior activity of ASN004 was demonstrated against trastuzumab-DM1, in a low-5T4/high-HER2 expressing gastric tumor model, and 10-fold greater potency was found for ASN004 against the 5T4-targeted ADC PF-06263507 in a lung tumor model. In marmoset monkeys, ASN004 was well tolerated at doses up to 1.5 mg/kg Q3W i.v., and showed dose-dependent exposure, linear pharmacokinetics, and markedly low exposure of free payload drug. Taken together, these findings identify ASN004 as a promising new ADC therapeutic for clinical evaluation in a broad range of solid tumor types.

Detection of Borrelia burgdorferi Cell-free DNA in Human Plasma Samples for Improved Diagnosis of Early Lyme Borreliosis.

BACKGROUND: Laboratory confirmation of early Lyme borreliosis (LB) is challenging. Serology is insensitive during the first days to weeks of infection, and blood polymerase chain reaction (PCR) offers similarly poor performance. Here, we demonstrate that detection of Borrelia burgdorferi (B.b.) cell-free DNA (cfDNA) in plasma can improve diagnosis of early LB. METHODS: B.b. detection in plasma samples using unbiased metagenomic cfDNA sequencing performed by a commercial laboratory (Karius Inc) was compared with serology and blood PCR in 40 patients with physician-diagnosed erythema migrans (EM), 28 of whom were confirmed to have LB by skin biopsy culture (n = 18), seroconversion (n = 2), or both (n = 8). B.b. sequence analysis was performed using investigational detection thresholds, different from Karius' clinical test. RESULTS: B.b. cfDNA was detected in 18 of 28 patients (64%) with laboratory-confirmed EM. In comparison, sensitivity of acute-phase serology using modified 2-tiered testing (MTTT) was 50% (P = .45); sensitivity of blood PCR was 7% (P = .0002). Combining B.b. cfDNA detection and MTTT increased diagnostic sensitivity to 86%, significantly higher than either approach alone (P ≤ .04). B.b. cfDNA sequences matched precisely with strain-specific sequence generated from the same individual's cultured B.b. isolate. B.b. cfDNA was not observed at any level in plasma from 684 asymptomatic ambulatory individuals. Among 3000 hospitalized patients tested as part of clinical care, B.b. cfDNA was detected in only 2 individuals, both of whom had clinical presentations consistent with LB. CONCLUSIONS: This is the first report of B.b. cfDNA detection in early LB and a demonstration of potential diagnostic utility. The combination of B.b. cfDNA detection and acute-phase MTTT improves clinical sensitivity for diagnosis of early LB.

Evaluation of Modified 2-Tiered Serodiagnostic Testing Algorithms for Early Lyme Disease.

Background: The conventional 2-tiered serologic testing protocol for Lyme disease (LD), an enzyme immunoassay (EIA) followed by immunoglobulin M and immunoglobulin G Western blots, performs well in late-stage LD but is insensitive in patients with erythema migrans (EM), the most common manifestation of the illness. Western blots are also complex, difficult to interpret, and relatively expensive. In an effort to improve test performance and simplify testing in early LD, we evaluated several modified 2-tiered testing (MTTT) protocols, which use 2 assays designed as first-tier tests sequentially, without the need of Western blots. Methods: The MTTT protocols included (1) a whole-cell sonicate (WCS) EIA followed by a C6 EIA; (2) a WCS EIA followed by a VlsE chemiluminescence immunoassay (CLIA); and (3) a variable major protein-like sequence, expressed (VlsE) CLIA followed by a C6 EIA. Sensitivity was determined using serum from 55 patients with erythema migrans; specificity was determined using serum from 50 patients with other illnesses and 1227 healthy subjects. Results: Sensitivity of the various MTTT protocols in patients with acute erythema migrans ranged from 36% (95% confidence interval [CI], 25%-50%) to 54% (95% CI, 42%-67%), compared with 25% (95% CI, 16%-38%) using the conventional protocol (P = .003-0.3). Among control subjects, the 3 MTTT protocols were similarly specific (99.3%-99.5%) compared with conventional 2-tiered testing (99.5% specificity; P = .6-1.0). Conclusions: Although there were minor differences in sensitivity and specificity among MTTT protocols, each provides comparable or greater sensitivity in acute EM, and similar specificity compared with conventional 2-tiered testing, obviating the need for Western blots.

Health policy basics: physician quality reporting system.

The U.S. health care system is in the midst of transforming from a fee-for-service system to a value-based system that delivers high-quality and cost-effective care. Quality reporting programs and increasing transparency of performance are meant to encourage physicians and hospitals to invest in improving the delivery of care. In 2006, the Centers for Medicare & Medicaid Services implemented the Physician Quality Reporting System (PQRS). The PQRS is an incentive and penalty payment program for eligible professionals who report data on quality measures for covered professional services furnished to Medicare beneficiaries. The program gives eligible professionals the opportunity to assess the quality of care they are providing to their patients and compare their performance on a given measure with that of their peers. This article discusses the history of PQRS, the 2014 PQRS, and how it affects other quality programs.

Commentary by the American College of Physicians on the "joint principles: integrating behavioral health care into the patient-centered medical home".

Comments on the article "Joint principles: Integrating behavioral health care into the patient-centered medical home" (see record 2014-24217-011). The American College of Physicians (ACP) supports the intent of these "Joint Principles" for behavioral health care and agrees that the incorporation of behavioral health care within the patient-centered medical home model is incomplete. ACP believes that these principles should be labeled as guidelines, rather than Joint Principles. ACP is also concerned with the use of overgeneralizations within the document that do not include a cited evidence base.

Burden and viability of Borrelia burgdorferi in skin and joints of patients with erythema migrans or lyme arthritis.

OBJECTIVE: To determine the burden and viability of Borrelia burgdorferi in the skin and joints of patients with Lyme disease. METHODS: Standard and quantitative polymerase chain reaction (PCR) techniques were used to detect B burgdorferi DNA in skin samples from 90 patients with erythema migrans (EM) and in synovial fluid (SF) from 63 patients with Lyme arthritis (LA) and in synovial tissue from 9 patients. Quantitative PCR determinations of B burgdorferi DNA, messenger RNA (mRNA), and ribosomal RNA (rRNA) were made in 10 skin samples from EM patients and 11 SF samples from LA patients. RESULTS: Skin lesions in most patients with EM had positive PCR results for B burgdorferi DNA. In the majority of patients with LA, a late disease manifestation, PCR results in pretreatment SF samples were positive. In patients with antibiotic-refractory arthritis, positive PCR results persisted for as long as 11 months, but positive results in samples taken during the postantibiotic period did not correlate with relapse or with the subsequent duration of arthritis, and at synovectomy, all results of PCR of synovial tissue were negative. B burgdorferi mRNA, a marker of spirochetal viability, was detected in 8 of 10 skin samples from EM patients, but in none of 11 SF samples from LA patients, even when obtained prior to antibiotic administration. Moreover, the median ratio of spirochetal rRNA to DNA, a measure of ribosomal activity, was 160 in the 10 EM skin samples, but only 0.15 in the 3 LA SF samples with positive results. CONCLUSION: B burgdorferi in the skin lesions of EM patients were active and viable, whereas those in the SF of LA patients were moribund or dead at any time point. Thus, detection of B burgdorferi DNA in SF is not a reliable test of active joint infection in Lyme disease.

Preclinical anti-tumor activity of antibody-targeted chemotherapy with CMC-544 (inotuzumab ozogamicin), a CD22-specific immunoconjugate of calicheamicin, compared with non-targeted combination chemotherapy with CVP or CHOP.

PURPOSE: CMC-544 (inotuzumab ozogamicin) is a CD22-specific immunoconjugate of calicheamicin currently being evaluated in patients with non-Hodgkin's B-cell lymphoma (BCL). CHOP and CVP represent untargeted combination chemotherapy comprised of cyclophosphamide, vincristine and prednisone with or without doxorubicin, commonly used in the treatment of NHL. Here, we describe anti-tumor efficacy of CMC-544, CHOP or CVP against human BCL xenografts. METHODS: In vitro, human BCLs were cultured with CMC-544 or individual constituents of CHOP for inhibition of their growth. In vivo, immunocompromised mice with established BCL xenografts were administered CHOP, CVP or CMC-544 to monitor their survival and BCL growth. RESULTS: In vitro, CMC-544 was more potent in causing growth inhibition of various BCL than cyclophosphamide, doxorubicin, vincristine or dexamethasone. In vivo, treatment with CHOP or CVP inhibited growth of BCL xenografts for up to 40 days after which BCL relapsed. Tumor growth inhibition by CMC-544 (>100 days) lasted longer than that by CHOP or CVP. BCL xenografts that relapsed after the treatment with CHOP or CVP were far less responsive to CHOP or CVP re-treatment but regressed upon subsequent treatment with CMC-544. CVP could be co-administered with suboptimal doses of CMC-544, while CHOP could be administered on alternant days with CMC-544 to cause enhanced regression of established BCL xenografts. CONCLUSION: Preclinically, CMC-544 provides greater therapeutic benefit than CVP or CHOP against BCL xenografts. CMC-544 may also be co-administered with standard chemotherapeutic regimens in the treatment of B-NHL for superior anti-tumor activity.

Prospective study of serologic tests for lyme disease.

BACKGROUND: Tests to determine serum antibody levels-the 2-tier sonicate immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and Western blot method or the IgG of the variable major protein-like sequence-expressed (VlsE) sixth invariant region (C6) peptide ELISA method-are the major tests available for support of the diagnosis of Lyme disease. However, these tests have not been assessed prospectively. METHODS: We used these tests prospectively to determine serologic responses in 134 patients with various manifestations of Lyme disease, 89 patients with other illnesses (with or without a history of Lyme disease), and 136 healthy subjects from areas of endemicity and areas in which the infection was not endemic. RESULTS: With 2-tier tests and the C6 peptide ELISA, only approximately one-third of 76 patients with erythema migrans had results that were positive for IgM or IgG seroreactivity with Borrelia burgdorferi in acute-phase samples. During convalescence, 3-4 weeks later, almost two-thirds of patients had seroreactivity with the spirochete B. burgdorferi. The frequencies of seroreactivity were significantly greater among patients with spirochetal dissemination than they were among those who lacked evidence of disseminated disease. Of the 44 patients with Lyme disease who had neurologic, heart, or joint involvement, all had positive C6 peptide ELISA results, 42 had IgG responses with 2-tier tests, and 2 patients with facial palsy had only IgM responses. However, among the control groups, the IgG Western blot was slightly more specific than the C6 peptide ELISA. The differences between the 2 test systems (2-tier testing and C6 peptide ELISA) with respect to sensitivity and specificity were not statistically significant. CONCLUSIONS: Except in patients with erythema migrans, both test systems were sensitive for support of the diagnosis of Lyme disease. However, with current methods, 2-tier testing was associated with slightly better specificity.

Higher mRNA levels of chemokines and cytokines associated with macrophage activation in erythema migrans skin lesions in patients from the United States than in patients from Austria with Lyme borreliosis.

BACKGROUND: Erythema migrans (EM) is caused primarily by Borrelia afzelii in Europe and solely by Borrelia burgdorferi in the United States. B. burgdorferi infection in the United States has previously been associated with faster expansion of EM lesions and with more associated symptoms, compared with B. afzelii infection in Europe. However, reasons for these differences are not yet known. METHODS: We determined the Borrelia species infecting 67 US or Austrian patients with EM. The clinical pictures and chemokine and cytokine mRNA levels in lesional skin were then compared in the 19 B. burgdorferi-infected US patients and the 37 B. afzelii-infected Austrian patients, the 2 largest groups. RESULTS: The 19 B. burgdorferi-infected US patients had faster-expanding EM lesions and a median of 4 associated signs and symptoms, whereas the 37 B. afzelii-infected Austrian patients had slower-expanding lesions and usually did not experience associated symptoms. Compared with the EM lesions of B. afzelii-infected Austrian patients, those of B. burgdorferi-infected US patients had significantly higher mRNA levels of chemokines associated with activation of macrophages, including chemoattractants for neutrophils (CXCL1), macrophages (CCL3 and CCL4), and T helper 1 cells (CXCL9, CXCL10, and CXCL11). In addition, compared with the EM lesions of Austrian patients, the EM lesions of US patients tended to have higher mRNA levels of the macrophage-associated proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha, and they had significantly higher mRNA expression of the antiinflammatory cytokines interleukin 10 and transforming growth factor beta. CONCLUSIONS: The EM lesions of B. burgdorferi-infected US patients expanded faster, were associated with more symptoms, and had higher mRNA levels of macrophage-associated chemokines and cytokines than did the EM lesions of B. afzelii-infected Austrian patients.

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.

CD20-specific antibody-targeted chemotherapy of non-Hodgkin's B-cell lymphoma using calicheamicin-conjugated rituximab.

Tumor-targeted delivery of a potent cytotoxic agent, calicheamicin, using its immunoconjugates is a clinically validated therapeutic strategy. Rituximab is a human CD20-specific chimeric antibody extensively used in B-NHL therapy. We investigated whether conjugation to calicheamicin can improve the anti-tumor activity of rituximab against human B-cell lymphoma (BCL) xenografts in preclinical models. BCL cells were cultured with rituximab or its calicheamicin conjugates and their in vitro growth was monitored. BCL cells were injected s.c. to establish localized xenografts in nude mice or i.v. to establish disseminated BCL in severe combined immunodeficient (scid) mice. I.p. treatment with rituximab or its calicheamicin conjugates was initiated and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. Conjugation of calicheamicin to rituximab vastly enhanced its growth inhibitory activity against BCL in vitro. Conjugation to calicheamicin had no deleterious effect on the effector functional activity of rituximab. Calicheamicin conjugated to rituximab with an acid-labile linker exhibited greater anti-tumor activity against s.c. BCL xenografts and improved survival of mice with disseminated BCL over that of unconjugated rituximab. Anti-tumor activities of rituximab conjugated to calicheamicin via an acid-stable linker were similar to that of unconjugated rituximab. Superior anti-tumor efficacy exhibited by a calicheamicin immunoconjugate of rituximab with an acid-labile linker over that of rituximab demonstrates the therapeutic potential of CD20-specific antibody-targeted chemotherapy strategy in the treatment of B-NHL.

Biopharmaceutical drug discovery using novel protein scaffolds.

In recent years, biopharmaceutical drug products have become hugely successful. However, they are often complex molecules that are expensive to manufacture. Commercial needs for cost-effective therapies have therefore led to the development of novel protein scaffold technologies that are increasingly being used for biopharmaceutical drug discovery. Major new scaffolds include single-domain antibodies, small modular immunopharmaceuticals, tetranectins, AdNectins, A-domain proteins, lipocalins and ankyrin repeat proteins. These scaffolds offer low-cost alternatives to classical antibody therapeutic strategies and some have shown early clinical promise. Further progress in the field will permit the commercially successful development of sophisticated protein therapeutics against complex disease targets.

Borrelia burgdorferi genetic markers and disseminated disease in patients with early Lyme disease.

Three genetic markers of Borrelia burgdorferi have been associated with disseminated disease: the OspC type, the 16S-23S rRNA intergenic spacer type (RST), and vlsE. Here, we modified previous methods so as to identify the three markers by PCR and restriction fragment length polymorphism in parallel, analyzed B. burgdorferi isolates from erythema migrans (EM) skin lesions in 91 patients, and correlated the results with evidence of dissemination. OspC type A was found approximately twice as frequently in patients with disseminated disease, whereas type K was identified approximately twice as often in those without evidence of dissemination, but these trends were not statistically significant. The remaining seven types identified were found nearly equally in patients with or without evidence of dissemination. RST 1 strains were significantly associated with dissemination (P=0.03), whereas RST 2 and RST 3 strains tended to have an inverse association with this outcome. The vlsE gene was identified in all 91 cases, using primer sets specific for an N-terminal sequence of B. burgdorferi strain B31 (vlsEB31) or strain 297 (vlsE297), but neither marker was associated with dissemination. Specific combinations of the three genetic markers usually occurred together. OspC type A was always found with RST 1 and vlsEB31, type K was always identified with RST 2 and more often with vlsE297, and types E and I were almost always found with RST 3 and equally often with vlsEB31 and vlsE297. We conclude that B. burgdorferi strains vary in their capacity to disseminate, but almost all strains isolated from EM lesions sometimes caused disseminated disease.

Tumoricidal effect of calicheamicin immuno-conjugates using a passive targeting strategy.

Calicheamicin is a potent chemotherapeutic with a low therapeutic index that requires targeting to tumor cells for its use in the clinic. To treat acute myeloid leukemia, calicheamicin has been conjugated to an antibody that recognizes CD33 (gemtuzumab ozogamicin). The application range of this 'active' targeting strategy is limited since it depends on specific antigen expression by tumor cells. This limitation could be reduced by using an antigen-independent 'passive targeting' strategy for calicheamicin. 'Passive targeting' relies on the dysfunctional vasculature of a neoplastic tumor that allows enhanced retention of macromolecules. We studied the efficacy of calicheamicin conjugated to various carrier molecules: i.e. immunoglobulin, albumin or PEGylated Fc fragments. In nude mice, a conjugate of anti-CD33 and calicheamicin accumulates in human tumor xenografts in the absence of detectable amounts of targeting antigen. Passive targeting provided sufficient accumulation of this conjugate to inhibit tumor growth of 10 different CD33-negative xenograft models. This efficacy depended on the use of an acid-labile linker between antibody and calicheamicin. Substitution of immunoglobulin as a carrier with either albumin or PEGylated Fc reduced or eliminated the efficacy of the conjugate. The results showed that using 'non-specific' immunoglobulin for passive targeting of calicheamicin might be an effective mode of cancer therapy.

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.

B-cell depletion inhibits arthritis in a collagen-induced arthritis (CIA) model, but does not adversely affect humoral responses in a respiratory syncytial virus (RSV) vaccination model.

We report the development of a mouse B cell-depleting immunoconjugate (anti-CD22 monoclonal antibody [mAb] conjugated to calicheamicin) and its in vivo use to characterize the kinetics of CD22+ B-cell depletion and reconstitution in murine primary and secondary lymphoid tissues. The effect of B-cell depletion was further studied in a murine collagen-induced arthritis (CIA) model and a respiratory syncytial virus (RSV) vaccination model. Our results show that (1) the immunoconjugate has B-cell-specific in vitro and in vivo cytotoxicity; (2) B-cell reconstitution starts in the bone marrow and spleen around day 30 after depletion and is completed in all tissues tested by day 50; (3) B-cell depletion inhibits the development of clinical and histologic arthritis in the CIA model; (4) depletion of type II collagen antibody levels is not necessary for clinical and histologic prevention of CIA; and (5) B-cell depletion does not adversely affect memory antibody responses after challenge nor clearance of infectious virus from lungs in the RSV vaccination model. These results demonstrate for the first time that only B-cell reduction but not type II collagen antibody levels correlate with the prevention of arthritis and represent key insights into the role of CD22-targeted B-cell depletion in mouse autoimmunity and vaccination models.