Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 106 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19dim or CD19- B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22+ cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.

BACKGROUND

The prognosis for adults with relapsed acute lymphoblastic leukemia is poor. We sought to determine whether inotuzumab ozogamicin, an anti-CD22 antibody conjugated to calicheamicin, results in better outcomes in patients with relapsed or refractory acute lymphoblastic leukemia than does standard therapy.

METHODS

In this phase 3 trial, we randomly assigned adults with relapsed or refractory acute lymphoblastic leukemia to receive either inotuzumab ozogamicin (inotuzumab ozogamicin group) or standard intensive chemotherapy (standard-therapy group). The primary end points were complete remission (including complete remission with incomplete hematologic recovery) and overall survival.

RESULTS

Of the 326 patients who underwent randomization, the first 218 (109 in each group) were included in the primary intention-to-treat analysis of complete remission. The rate of complete remission was significantly higher in the inotuzumab ozogamicin group than in the standard-therapy group (80.7% [95% confidence interval {CI}, 72.1 to 87.7] vs. 29.4% [95% CI, 21.0 to 38.8], P<0.001). Among the patients who had complete remission, a higher percentage in the inotuzumab ozogamicin group had results below the threshold for minimal residual disease (0.01% marrow blasts) (78.4% vs. 28.1%, P<0.001); the duration of remission was longer in the inotuzumab ozogamicin group (median, 4.6 months [95% CI, 3.9 to 5.4] vs. 3.1 months [95% CI, 1.4 to 4.9]; hazard ratio, 0.55 [95% CI, 0.31 to 0.96]; P=0.03). In the survival analysis, which included all 326 patients, progression-free survival was significantly longer in the inotuzumab ozogamicin group (median, 5.0 months [95% CI, 3.7 to 5.6] vs. 1.8 months [95% CI, 1.5 to 2.2]; hazard ratio, 0.45 [97.5% CI, 0.34 to 0.61]; P<0.001); the median overall survival was 7.7 months (95% CI, 6.0 to 9.2) versus 6.7 months (95% CI, 4.9 to 8.3), and the hazard ratio was 0.77 (97.5% CI, 0.58 to 1.03) (P=0.04). In the safety population, the most frequent grade 3 or higher nonhematologic adverse events with inotuzumab ozogamicin were liver-related. Veno-occlusive liver disease of any grade occurred in 15 patients (11%) who received inotuzumab ozogamicin and in 1 patient (1%) who received standard therapy.

CONCLUSIONS

The rate of complete remission was higher with inotuzumab ozogamicin than with standard therapy, and a higher percentage of patients in the inotuzumab ozogamicin group had results below the threshold for minimal residual disease. Both progression-free and overall survival were longer with inotuzumab ozogamicin. Veno-occlusive liver disease was a major adverse event associated with inotuzumab ozogamicin. (Funded by Pfizer; INO-VATE ALL ClinicalTrials.gov number, NCT01564784.).

The sialic acids are acidic monosaccharides typically found at the outermost ends of the sugar chains of animal glycoconjugates. They potentially can inhibit intermolecular and intercellular interactions by virtue of their negative charge. However, they can also act as critical components of ligands recognized by a variety of proteins of animal, plant, and microbial origin (sialic acid binding lectins). Recognition can be affected by specific structural variations and modifications of sialic acids, their linkage to the underlying sugar chain, the structure of these chains, and the nature of the glycoconjugate to which they are attached. Presented here is a summary of the various proteins that can recognize and bind to this family of monosaccharides, comparing and contrasting the structural requirements and mechanisms involved in binding. Particular attention is focused on the recently evolving information about sialic acid recognition by certain C-type lectins (the selectins), I-type lectins (e.g., CD22 and sialoadhesin), and a complement regulatory protein (the H protein). The last two instances are examples of the importance of the side chain of sialic acids and the effects of natural substitutions (e.g., 9-O-acetylation) of this part of the molecule.

CD22 is a membrane immunoglobulin (mIg)-associated protein of B cells. CD22 is tyrosine-phosphorylated when mIg is ligated. Tyrosine-phosphorylated CD22 binds and activates SHP, a protein tyrosine phosphatase known to negatively regulate signaling through mIg. Ligation of CD22 to prevent its coaggregation with mIg lowers the threshold at which mIg activates the B cell by a factor of 100. In secondary lymphoid organs, CD22 may be sequestered away from mIg through interactions with counterreceptors on T cells. Thus, CD22 is a molecular switch for SHP that may bias mIg signaling to anatomic sites rich in T cells.

BACKGROUND

Hairy-cell leukemia that is resistant to treatment with purine analogues, including cladribine, has a poor prognosis. We tested the safety and efficacy of an immunotoxin directed against a surface antigen that is strongly expressed by leukemic hairy cells.

METHODS

RFB4(dsFv)-PE38 (BL22), a recombinant immunotoxin containing an anti-CD22 variable domain (Fv) fused to truncated pseudomonas exotoxin, was administered in a dose-escalation trial by intravenous infusion every other day for a total of three doses.

RESULTS

Of 16 patients who were resistant to cladribine, 11 had a complete remission and 2 had a partial remission with BL22. The three patients who did not have a response received low doses of BL22 or had preexisting toxin-neutralizing antibodies. Of the 11 patients in complete remission, 2 had minimal residual disease in the bone marrow or blood. During a median follow-up of 16 months (range, 10 to 23), 3 of the 11 patients who had a complete response relapsed and were retreated; all of these patients had a second complete remission. In 2 of the 16 patients, a serious but completely reversible hemolytic-uremic syndrome developed during the second cycle of treatment with BL22. Common toxic effects included transient hypoalbuminemia and elevated aminotransferase levels.

CONCLUSIONS

BL22 can induce complete remissions in patients with hairy-cell leukemia that is resistant to treatment with purine analogues.

CD22 is a surface glycoprotein of B lymphocytes that is rapidly phosphorylated on cytoplasmic tyrosines after antigen receptor cross-linking. Splenic B cells from mice with a disrupted CD22 gene were found to be hyperresponsive to receptor signaling: Heightened calcium fluxes and cell proliferation were obtained at lower ligand concentrations. The mice gave an augmented immune response, had an expanded peritoneal B-1 cell population, and contained increased serum titers of autoantibody. Thus, CD22 is a negative regulator of antigen receptor signaling whose onset of expression at the mature B cell stage may serve to raise the antigen concentration threshold required for B cell triggering.

BACKGROUND

. Antibody responses are triggered by binding of antigen to the B-cell antigen receptor (BCR). The strength of the resulting signal determines the outcome of the response, which may vary from the induction of tolerance to the antigen, to the production of specific high-affinity antibodies. Additional cell-surface proteins assist the BCR in its function, and can facilitate or inhibit an antibody response. CD22 is a BCR-associated transmembrane protein, the cytoplasmic tail of which contains three immunoreceptor tyrosine-based inhibitory motifs. These motifs are phosphorylated upon BCR-crosslinking, and can bind the tyrosine phosphatase SHP-1, a putative negative regulator of signalling from the BCR. In order to assess the role of CD22 in vivo, we have generated CD22(-/-) mice by targeted gene inactivation.

RESULTS

. In CD22(-/-) mice, B-cell development is normal. There are normal numbers of peripheral B cells, but these have a more mature phenotype. In addition, recirculating B cells are absent from the bone marrow. However, the distribution of the two B-cell subtypes, B-1 and B-2, is normal. After BCR-crosslinking in vitro, splenic CD22(-/-) B cells show an increased Ca2+ influx and a lower survival due to an increased induction of apoptosis. In contrast, there is an increased proliferative response to the B-cell mitogen lipopolysaccharide (LPS). A shorter average lifespan in the B-cell compartment is also found in vivo. Furthermore, T-cell independent immune responses are impaired, whereas T-cell dependent responses are normal.

CONCLUSIONS

. The absence of CD22 expression lowers the signalling threshold for BCR-crosslinking and can thus influence the fate of the B cell. We propose that the low threshold leads to hyperresponsiveness of the B cells and a chronic basal activation. In this model, engagement of the receptor without T-cell help leads to an increased induction of apoptosis, thus explaining the shorter lifespan of CD22(-/-) B cells and the low response to T-cell independent antigens. The alteration in B-cell phenotype and the higher levels of LPS-reactivity are attributable to the chronic basal stimulation.

Estrogen is thought to contribute to the increased frequency of autoimmune disorders occurring in females, but a molecular basis for its effects on autoimmunity remains to be elucidated. We have shown previously that estrogen leads to the survival and activation of autoreactive cells in the naive repertoire. To identify the molecular pathways involved in B cell tolerance, we sought to identify genes that are differentially regulated by estrogen in mouse B cells. Several genes involved in B cell activation and survival, including cd22, shp-1, bcl-2, and vcam-1, were upregulated by estrogen in B cells. We found that overexpression of CD22 and SHP-1 in B cells decreased B cell receptor signaling. Estrogen receptors alpha and beta are expressed on B cells and are functional, since they can directly upregulate expression of CD22, SHP-1, and Bcl-2. Estrogen treatment protected isolated primary B cells from B cell receptor-mediated apoptosis. These results suggest that estrogen induces a genetic program that alters survival and activation of B cells in a B cell-autonomous fashion and thus skews the naive immune system toward autoreactivity.

OBJECTIVE

To conduct a phase I dose-escalation trial assessing safety and response of recombinant immunotoxin moxetumomab pasudotox (CAT-8015, HA22) in chemotherapy-resistant hairy cell leukemia (HCL).

METHODS

Eligible patients had relapsed/refractory HCL after ≥ two prior therapies and required treatment because of abnormal blood counts. Patients received moxetumomab pasudotox 5 to 50 μg/kg every other day for three doses (QOD ×3), with up to 16 cycles repeating at ≥ 4-week intervals if patients did not experience disease progression or develop neutralizing antibodies.

RESULTS

Twenty-eight patients were enrolled, including three patients each at 5, 10, 20, and 30 μg/kg, four patients at 40 μg/kg, and 12 patients at 50 μg/kg QOD ×3 for one to 16 cycles each (median, four cycles). Dose-limiting toxicity was not observed. Two patients had transient laboratory abnormalities consistent with grade 2 hemolytic uremic syndrome with peak creatinine of 1.53 to 1.66 mg/dL and platelet nadir of 106,000 to 120,000/μL. Drug-related toxicities in 25% to 64% of the 28 patients included (in decreasing frequency) grade 1 to 2 hypoalbuminemia, aminotransferase elevations, edema, headache, hypotension, nausea, and fatigue. Of 26 patients evaluable for immunogenicity, 10 patients (38%) made antibodies neutralizing more than 75% of the cytotoxicity of 1,000 ng/mL of immunotoxin, but this immunogenicity was rare (5%) after cycle 1. The overall response rate was 86%, with responses observed at all dose levels, and 13 patients (46%) achieved complete remission (CR). Only 1 CR lasted less than 1 year, with the median disease-free survival time not yet reached at 26 months.

CONCLUSIONS

Moxetumomab pasudotox at doses up to 50 μg/kg QOD ×3 has activity in relapsed/refractory HCL and has a safety profile that supports further clinical development for treatment of this disease.

The ST6Gal sialyltransferase controls production of the Siaalpha2-6Galbeta1-4GlcNAc (Sia6LacNAc) trisaccharide, which is the ligand for the lectin CD22. Binding of CD22 to Sia6LacNAc is implicated in regulating lymphocyte adhesion and activation. We have investigated mice that lack ST6Gal and report that they are viable, yet exhibit hallmarks of severe immunosuppression unlike CD22-deficient mice. Notably, Sia6LacNAc-deficient mice display reduced serum IgM levels, impaired B cell proliferation in response to IgM and CD40 crosslinking, and attenuated antibody production to T-independent and T-dependent antigens. Deficiency of ST6Gal was further found to alter phosphotyrosine accumulation during signal transduction from the B lymphocyte antigen receptor. These studies reveal that the ST6Gal sialyltransferase and corresponding production of the Sia6LacNAc oligosaccharide are essential in promoting B lymphocyte activation and immune function.

A B lymphocyte hyperactivity syndrome resembling systemic lupus erythematosus characterizes mice lacking the src-family kinase Lyn. Lyn is not required to initiate B cell antigen receptor (BCR) signaling but is an essential inhibitory component. lyn-/- B cells have a delayed but increased calcium flux and exaggerated negative selection responses in the presence of antigen and spontaneous hyperactivity in the absence of antigen. As in invertebrates, genetic effects of loci with only one functional allele can be used to analyze signaling networks in mice, demonstrating that negative regulation of the BCR is a complex quantitative trait in which Lyn, the coreceptor CD22, and the tyrosine phosphatase SHP-1 are each limiting elements. The biochemical basis of this complex trait involves a pathway requiring Lyn to phosphorylate CD22 and recruit SHP-1 to the CD22/BCR complex.

B cell activation following antigen receptor cross-linking can be augmented in vitro by ligation of cell surface CD22, which associates with the SHP1 protein tyrosine phosphatase. The targeted deletion of CD22 in mice demonstrated that CD22 differentially regulates antigen receptor signaling in resting and antigen-stimulated B lymphocytes. B cells from CD22-deficient mice exhibited the cell surface phenotype and augmented intracellular calcium responses characteristic of chronically stimulated B cells, as occurs in SHP1-defective mice. Thus, CD22 negatively regulates antigen receptor signaling in the absence of antigen. However, activation of CD22-deficient B lymphocytes by prolonged IgM cross-linking resulted in modest B cell proliferation, demonstrating that CD22 positively regulates antigen receptor signaling in the presence of antigen.

We have investigated the role of immunophenotyping in distinguishing between leukemic B-cell lymphoproliferative disorders. Circulating cells from 666 cases were analyzed with a panel of markers by flow cytometry. The diseases included: chronic lymphocytic leukemia (CLL), 400; prolymphocytic leukemia, 22; hairy cell leukemia (HCL), 40; HCL variant, 15; splenic lymphoma with villous lymphocytes, 100; follicular lymphoma, 26; lymphoplasmacytic lymphoma, 25; mantle-cell lymphoma, 20; and large cell lymphoma, 18. On the basis of the most common marker profile in CLL, CD5+, CD23+, FMC7- and weak expression (+/-) of surface immunoglobulin (SmIg) and CD22, we devised a scoring system that gives for each of these five markers a value of 1 or 0 according to whether it is typical or atypical for CLL. Scores range from 5 (typical of CLL) to 0 (atypical for CLL). Application of the scoring system to all the cases showed that 87% of CLL scored 5 and 4 and only 0.4% scored 0 or 1, whereas 89% of other B-cell leukemias and 72% of lymphomas scored 0 or 1; only one case (0.3%) scored 4 and none scored 5 (p < 0.0001). There were no differences between CLL with high and low scores but higher scores were found in cases with more typical morphology (p < 0.0015). Considering each individual marker, there was no single one that distinguished CLL from other diseases, although the most reliable were SmIg intensity and FMC7. The proposed score will facilitate the diagnosis of B-lymphoproliferative disorders and improve their classification.

OBJECTIVE

To conduct a phase I trial of recombinant immunotoxin BL22, an anti-CD22 Fv fragment fused to truncated Pseudomonas exotoxin.

METHODS

Forty-six pretreated patients with CD22+ non-Hodgkin's lymphoma (NHL; n = 4), chronic lymphocytic leukemia (CLL; n = 11), and hairy cell leukemia (HCL; n = 31) received 265 cycles at 3 to 50 microg/Kg every other day x 3 doses.

RESULTS

BL22 was active in HCL, with 19 complete remissions (CRs; 61%) and six partial responses (PRs; 19%) in 31 patients. Of 19 CRs, 11 were achieved after one cycle and eight after two to 14 cycles. All 25 responders benefited clinically with one cycle. The CR rate was 86% in patients enrolled at>> or = 40 microg/Kg every other day x 3, and 41% at lower doses (P = .011). The median duration for CR was 36 months (range, 5 to 66 months), and eight patients remain in CR at 45 months (range, 29 to 66 months). Lower but significant activity occurred in CLL. Neutralizing antibodies occurred in 11 (24%) of 46 patients (all HCL). A reversible hemolytic uremic syndrome requiring plasmapheresis was observed in one patient with NHL during cycle 1 and in four patients with HCL during cycle 2 or 3. The maximum-tolerated dose (MTD) evaluated at cycle 1 was 40 microg/Kg IV. QOD x 3. The most common toxicities at 30 to 50 microg/Kg every other day x 3 included hypoalbuminemia, transaminase elevations, fatigue, and edema.

CONCLUSIONS

BL22 was well tolerated and highly effective in HCL, even after one cycle. Phase II testing is underway to define the efficacy with one cycle and to study safety when additional cycles are needed for optimal response.

BACKGROUND

The outlook for patients with refractory and relapsed acute lymphocytic leukaemia (ALL) is poor. CD22 is highly expressed in patients with ALL. Inotuzumab ozogamicin is a CD22 monoclonal antibody conjugated to the toxin calecheamicin. We did a phase 2 study to assess the efficacy of this antibody.

METHODS

We recruited patients at the MD Anderson Cancer Center, Houston, TX, USA, between June, 2010, and March, 2011. Adults and children with refractory and relapsed ALL were eligible. Ten adults were treated before enrolment of children started. Patients were given 1·8 mg/m(2) inotuzumab ozogamicin intravenously over 1 h every 3-4 weeks (the first three adults and three children received 1·3 mg/m(2) in the first course). The primary endpoint was overall response (complete response or marrow complete response with no recovery of platelet count or incomplete recovery of neutrophil and platelet counts). Analysis was done by intention to treat. This study is registered, number NCT01134575.

RESULTS

49 patients were enrolled and treated. Median age was 36 years (range 6-80). CD22 was expressed in more than 50% of blasts in all patients. The median number of courses was two (range one to five) and the median time between courses was 3 weeks (range 3-6). Nine (18%) patients had complete response, 19 (39%) had marrow complete response, 19 (39%) had resistant disease, and two (4%) died within 4 weeks of starting treatment. The overall response rate was 57% (95% CI 42-71). The most frequent adverse events during course one of treatment were fever (grade 1-2 in 20 patients, grade 3-4 in nine), hypotension (grade 1-2 in 12 patients, grade 3 in one), and liver-related toxic effects (bilirubin: grade 1-2 in 12 patients, grade 3 in two; raised aminotransferase concentration: grade 1-2 in 27 patients, grade 3 in one).

CONCLUSIONS

Inotuzumab ozogamicin shows promise as a treatment for refractory and relapsed ALL.

BACKGROUND

Pfizer.

OBJECTIVE

To verify the hypothesis that in rheumatoid arthritis (RA), tumor necrosis factor alpha (TNFalpha) plays a critical role in regulating leukocyte trafficking and chemokine levels.

METHODS

Ten patients with longstanding RA received a single 10 mg/kg infusion of anti-TNFalpha monoclonal antibody (cA2). The articular localization of autologous granulocytes, separated in vitro and labeled with 111In, was studied by analysis of gamma-camera images both before and 2 weeks after treatment. At the same sequential time points, synovial biopsy samples were assessed for infiltrating CD3+ T cells, CD22+ B cells, and CD68+ macrophages. Synovial tissue expression of the chemokines interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, Groalpha, and RANTES was also determined. Serum IL-8 and MCP-1 concentrations were measured by enzyme-linked immunosorbent assay.

RESULTS

Anti-TNFalpha therapy in RA significantly reduced 111In-labeled granulocyte migration into affected joints. There was a simultaneous and significant reduction in the numbers of infiltrating synovial CD3+ T cells, CD22+ B cells, and CD68+ macrophages and in the expression of IL-8 and MCP-1, with a trend toward a reduction in serum concentrations of these chemokines.

CONCLUSIONS

TNFalpha blockade reduces synovial expression of the chemokines IL-8 and MCP-1 and diminishes inflammatory cell migration into RA joints.

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.

Immunotoxins are proteins that contain a toxin along with an antibody or growth factor that binds specifically to target cells. Nearly all protein toxins work by enzymatically inhibiting protein synthesis. For the immunotoxin to work, it must bind to and be internalized by the target cells, and the enzymatic fragment of the toxin must translocate to the cytosol. Once in the cytosol, 1 molecule is capable of killing a cell, making immunotoxins some of the most potent killing agents. Various plant and bacterial toxins have been genetically fused or chemically conjugated to ligands that bind to cancer cells. Among the most active clinically are those that bind to hematologic tumors. At present, only 1 agent, which contains human interleukin-2 and truncated diphtheria toxin, is approved for use in cutaneous T-cell lymphoma. Another, containing an anti-CD22 Fv and truncated Pseudomonas exotoxin, has induced complete remissions in a high proportion of cases of hairy-cell leukemia. Refinement of existing immunotoxins and development of new immunotoxins are underway to improve the treatment of cancer.