CD22 ligand-binding and signaling domains reciprocally regulate B-cell Ca2+ signaling

Abstract Abstract 425 Background: Over 400,000 people in the United States are living with non-Hodgkin's lymphoma (NHL). While survival rates have improved, over 20,000 persons die annually from NHL. Cytotoxic chemotherapies are initially effective, but resistance can develop and dose limiting toxicities are problematic. Monoclonal antibody (mAb) therapy with rituximab, a chimeric anti-CD20 mAb, has shown benefit alone and in combination with chemotherapy, and can improve overall survival. New mAbs are being tested in combination with rituximab, including bispecific antibodies (BsAb) that simultaneously target CD20 and CD22. CD22 is a B-lymphocyte specific adhesion molecule expressed on nearly all mature B-cells, including the majority of B cell-NHLs. HB22.7 is an anti-CD22 mAb that specifically blocks the interaction of CD22 with its ligand, initiates CD22-mediated signal transduction, and has direct cytotoxic effects. Anti-CD22 mAbs that do not block ligand binding possess only modest functional effects; prior BsAbs contained non-ligand binding anti-CD22 mAbs. Therefore, we constructed a BsAb (Bs20x22) using rituximab and HB22.7, and evaluated its cell binding, signaling patterns, and lymphomacidal activity using a human NHL xenograft model. Methods: Bs20x22 was constructed from F(ab')2 fragments of rituximab and HB22.7 using the ImmunoPure F(ab')2 Preparation Kit (Pierce). Cell binding studies used the CD20/CD22 double positive human Burkitt's B-cell lymphoma lines Raji and Ramos, and the CD20/CD22 double negative human embryonic kidney cell line, 293T. In vitro cytotoxicity was assessed by trypan blue exclusion. In vitro apoptosis assays were performed on plated Raji or Ramos cells which were then treated with HB22.7, rituximab, HB22.7 plus rituximab, or Bs20x22. The total number of cells and the number of apoptotic cells were counted, with results expressed as the % of control (% apoptotic cells treated / % apoptotic cells untreated). Results: Bs20x22 specifically bound CD20 and CD22, similar to the parent mAbs rituximab and HB22.7. In vitro cytotoxicity assays showed that Bs20x22 was three times more effective than either parent mAb alone and twice as effective as a combination of both parent mAb used at equimolar concentrations. Additionally, Bs20x22 was nearly four times more effective at inducing apoptosis than either mAb alone, with the percentage of apoptotic cells greatest for Bs20x22 treatment (20.1%), compared to combination rituximab plus HB22.7 (7.5%), rituximab (6.7%), and HB22.7 (6.5%). Examination of the MAPK and SAPK signaling cascades revealed that treatment with Bs20x22 resulted in significant activation of p38, while treatment with either parent mAb did not. In an in vivo human NHL xenograft model, treatment with Bs20x22 resulted in significantly greater tumor shrinkage resulting in the smallest tumor volume of any group. Bs20x22 treated mice had the best survival rate (88%) compared to the combination of rituximab plus HB22.7 (75%), rituximab (50%), HB22.7 (25%), and PBS control (0%). Significantly greater efficacy was also found when Bs20x22 was administered prior to the development of visible tumors versus treatment of established tumors, with tumors 89% smaller in mice treated pre-emptively. Conclusions: This study demonstrates the feasibility of constructing a bispecific antibody targeting both CD20 and CD22, using an anti-CD22 mAb with ligand-blocking ability. Efficacy was demonstrated by in vitro cytotoxicity and apoptosis assays, p38 activation, and human NHL xenograft models. Our results suggest that Bs20x22 is more efficacious than the combination of rituximab and HB22.7, and use of Bs20x22 eliminates the need for sequential administration of two separate mAbs. Disclosures: No relevant conflicts of interest to declare.

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Pilot study of redirected autologous T cells engineered to contain anti-CD19 attached to TCRζ and 4-1BB signaling domains in patients with chemotherapy-resistant or -refractory CD19+ leukemia and lymphoma.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.tps10072 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. TPS10072-TPS10072

Author(s):

Stephan A. Grupp ◽

David L. Porter ◽

Bruce Levine ◽

Michael Kalos ◽

Christine Strait ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

T Cell Activation ◽

Chimeric Antigen Receptor ◽

Intracellular Signaling ◽

Lymphoblastic Leukemia ◽

Antigen Receptor ◽

Study Cohort ◽

B Cell Malignancies ◽

Signaling Domains

TPS10072 Background: Outcomes remain poor for patients (pts) with relapsed or refractory (r/r) B-cell malignancies such as acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). CD19 is an attractive therapeutic target because it is widely expressed on normal and malignant B cells throughout B-cell maturation but not on pluripotent stem cells or non–B-cell tissues. We have developed chimeric antigen receptor T cells to target CD19+ cells (CART-19 or CTL019). This approach involves patient-derived T cells that are genetically modified via lentiviral transduction to express a CD19 antigen recognition domain attached to intracellular signaling domains (TCRζ and 4-1BB) that mediate T-cell activation. A recent study in CLL showed that CART-19 therapy had potent activity with responses in 5/8 evaluable pts (3 CR, 2 PR). Pts achieving CR (two > 24 months and one > 5 months) remain in CR with detectable CART-19 cells (Porter et al. ASH 2012). Here we describe a study of CART-19 therapy in pediatric pts with r/r leukemia and lymphoma (NCT01626495). Methods: Pts eligible for this single-arm, open-label study are aged 1 to 24 years with r/r CD19+ B-cell malignancies, without a transplant option or having relapsed after allogeneic stem cell transplantation (ASCT). Pts will undergo leukapheresis to obtain T cells, which will be stimulated, expanded, and transduced ex vivo to express the chimeric antigen receptor. Pts may receive lymphodepleting chemotherapy prior to CART-19 infusion. Study objectives: determine the safety and feasibility of administering CART-19 therapy to pediatric pts, assess duration of in vivo survival of CART-19 cells, and measure antitumor response. There are 2 cohorts in the study: Cohort 1 includes pts who have not undergone ASCT and are not currently eligible for a transplant, and cohort 2 includes pts who relapsed after ASCT. Cells are collected from the recipient, not the donor, which allows for this approach to be used in pts s/p cord blood transplant, and which we hypothesize will reduce the risk of GVHD as a result of toleration of the T cells in the recipient. To date, 7 pts have been enrolled. Clinical trial information: NCT01626495.

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The Ligand-binding Domain of CD22 Is Needed for Inhibition of the B Cell Receptor Signal, as Demonstrated by a Novel Human CD22-specific Inhibitor Compound

Journal of Experimental Medicine ◽

10.1084/jem.20011783 ◽

2002 ◽

Vol 195 (9) ◽

pp. 1207-1213 ◽

Cited By ~ 127

Author(s):

Soerge Kelm ◽

Judith Gerlach ◽

Reinhard Brossmer ◽

Claus-Peter Danzer ◽

Lars Nitschke

Keyword(s):

B Cells ◽

Cell Surface ◽

Ligand Binding ◽

B Cell ◽

Intracellular Signaling ◽

Cell Receptor ◽

B Cell Receptor ◽

Negative Regulator ◽

Target Cells ◽

Lectin Domain

CD22 is a B cell–specific transmembrane protein of the Siglec family. It binds specifically to α2,6-linked sialic acid (Sia) residues, which are also present on glycoproteins on the B cell surface. CD22 acts as a negative regulator in B cell receptor–mediated signaling by recruitment of Src homology 2 domain–containing tyrosine phosphatase (SHP)-1 to its intracellular tail. To analyze how ligand-binding of CD22 influences its intracellular signaling domain, we designed synthetic sialosides as inhibitors for the lectin domain of CD22. One of these compounds inhibited binding of human CD22-Fc to target cells over 200-fold better than Sia and was highly selective for human CD22. When Daudi cells or primary B cells were stimulated with anti-immunoglobulin (Ig)M in presence of this sialoside inhibitor, a higher Ca2+ response was observed, similar to CD22-deficient B cells. Accordingly, a lower tyrosine-phosphorylation of CD22 and SHP-1 recruitment was demonstrated in presence of the sialoside. Thus, by interfering with ligand binding of CD22 on the B cell surface, we have shown for the first time that the lectin domain of CD22 has a direct, positive influence on its intracellular inhibitory domain. Also, we have developed a novel low molecular weight compound which can enhance the response of human B cells.

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Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex

Blood ◽

10.1182/blood-2012-12-471094 ◽

2013 ◽

Vol 121 (23) ◽

pp. 4729-4739 ◽

Cited By ~ 34

Author(s):

Jillian F. Wise ◽

Zuzana Berkova ◽

Rohit Mathur ◽

Haifeng Zhu ◽

Frank K. Braun ◽

...

Keyword(s):

Ligand Binding ◽

B Cell ◽

Fas Ligand ◽

B Cell Lymphomas ◽

Key Points ◽

Surface Nucleolin

Key Points B-cell lymphomas with surface nucleolin-Fas complexes are resistant to Fas-mediated apoptosis through decreased ligand binding. Expression of nucleolin protects mice from a lethal agonistic Fas challenge, whereas a non-Fas binding nucleolin mutant does not.

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A New High Activity Anti-CD22 Chimeric Antigen Receptor (CAR) Targeting B Cell Leukemia.

Blood ◽

10.1182/blood.v120.21.2611.2611 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2611-2611

Author(s):

Waleed Haso ◽

Daniel W. Lee ◽

Ira Pastan ◽

Dimiter S. Dimitrov ◽

Crystal L Mackall ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Cell Lines ◽

Second Generation ◽

Third Generation ◽

Cytokine Release ◽

Binding Motifs ◽

Signaling Domains

Abstract Abstract 2611 CD22 is expressed on the surface of B cell hematologic malignancies such as acute lymphoblastic leukemia (ALL). CD22 is a Siglec family lectin present on B cells, starting at the pre-B cell stage of development, but is not expressed on plasma cells. CD22 consists of 7 extracellular Ig domains and is found in 2 isoforms, one of which is missing the second and third N-terminal Ig domains. We generated CAR modified T cells containing anti-CD22 extracellular binding motifs fused to intracellular signaling domains for T cells activation (CD3 zeta) or costimulation (CD28 or 4-1BB). The binding motifs were derived from scFvs that targeted a membrane distal epitope of CD22, Ig domain 3, (BL22 and a higher affinity HA22 motif) or that bound a more membrane proximal, Ig domains 5–7, of CD22 (m971). The CAR constructs we generated were second-generation (CD28 and CD3 zeta; or, 4-1BB and CD3 zeta) or third generation (CD28, 4-1BB and CD3 zeta signaling domains). A CH2CH3 spacer domain from IgG1 was added in some constructs to examine the impact of extending the scFv-derived binding domain away from the transduced T cell membrane. In vitro cellular cytotoxicity and cytokine release experiments with 4 B cell-ALL cell lines (REH, SEM, NALM6, KOPN8) as well as the CD22 (+)ve Daudi and Raji cell lines were performed. Our results demonstrate that addition of the CH2CH3 domain did not improve tumor lysis and that standard affinity BL22 and higher affinity HA22-derived scFv epitopes were equivalent. With regard to signaling domains, second generation constructs were better than third generation constructs both in vitro and in vivo. In comparison between second generation constructs, CD28 containing domains outperformed 4-1BB with regard to lytic activity and cytokine release. Most surprising was the activity of the m971-derived scFv binding epitope. m971-CAR had significantly higher killing activity, a far more robust cytokine release profile, and superior in vivo activity. NSG mice were injected i.v. with 0.5× 106 NALM6-GL cells (pre-B cell ALL line engineered to express luciferase). Three days later, when disease was evident, mice were treated with 1×107 CAR+ T cells, and then followed by bioluminescent imaging to measure disease burden. The m971 CAR was significantly more potent at tumor clearance than our previously developed most active construct expressing the HA22-derived scFv domain (Figure 1). Disease progressed rapidly when non-transduced T cells were used (mock). We are currently examining the activity of different signaling domains on m971 CAR efficacy in vivo and directly comparing the anti-CD22 m971 CAR to the CD19 CAR currently being evaluated in clinical trials. These studies will guide future anti-CD22 CAR-based anti-leukemia immunotherapy trials. Disclosures: No relevant conflicts of interest to declare.

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Ligand binding sites for a synthetic B cell growth and differentiation factor

Cellular Immunology ◽

10.1016/0008-8749(89)90301-8 ◽

1989 ◽

Vol 123 (2) ◽

pp. 417-426 ◽

Cited By ~ 7

Author(s):

Michael G. Goodman ◽

Dana M. Cherry

Keyword(s):

Cell Growth ◽

Ligand Binding ◽

B Cell ◽

Binding Sites ◽

Differentiation Factor ◽

Ligand Binding Sites ◽

Cell Growth And Differentiation

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Anti-IgM-mediated B cell signaling. Molecular analysis of ligand binding requisites for human B cell clonal expansion and tolerance.

Journal of Experimental Medicine ◽

10.1084/jem.168.1.247 ◽

1988 ◽

Vol 168 (1) ◽

pp. 247-266 ◽

Cited By ~ 28

Author(s):

S M Rudich ◽

K H Roux ◽

R J Winchester ◽

P K Mongini

Keyword(s):

Signal Transduction ◽

Ligand Binding ◽

Dna Synthesis ◽

B Cell ◽

Clonal Expansion ◽

Negative Feedback Regulation ◽

Inhibitory Signaling ◽

The Impact ◽

Cell Clonal Expansion ◽

Human B Cell

The ligand binding requisites for membrane IgM-mediated signaling of human B lymphocyte clonal expansion and B cell tolerance were investigated with a well-characterized set of soluble murine anti-human IgM mAbs. Evaluation of the impact of mu chain domain specificity, affinity, and binding stoichiometry for membrane IgM on antibody-induced regulation of normal and leukemic B cell DNA synthesis revealed that the ligand binding requisites for inducing or, alternatively, suppressing B cell DNA synthesis are significantly different. First, while the induction of S phase entry required micrograms/ml concentrations of ligand, orders of magnitude lower concentrations of ligand sufficed for inhibitory signaling. Second, while an upper affinity threshold for achieving maximal stimulation of B cell DNA synthesis was never detected, inhibitory signaling by bivalent ligands appeared to become relatively affinity independent at Fab binding affinities greater than 7.0 x 10(6) M-1. Third, while a C mu 1-specific mAb with an enhanced incidence of monogamous binding to mIgM was ineffective at inducing B cell DNA synthesis, the antibody was not significantly compromised in ability to initiate inhibitory signals. These differences could be observed in a clonal B cell population which positively or negatively responded to mIgM ligation depending upon its state of activation. The accumulated observations indicate that the ligand binding requisites for inhibitory signal transduction in human B lymphocytes are much less rigorous than those for stimulatory signal transduction and suggest that many physiologically relevant anti-Ig antibodies are more likely to function in the negative feedback regulation of B cell responses than in the direct triggering of human B cell clonal expansion.

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A Novel Candidate Gene for Predisposition of Hodgkin Lymphoma; Pregnan X Receptor (PXR)

Blood ◽

10.1182/blood-2021-149824 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4490-4490

Author(s):

Rejin Kebudi ◽

Yücel Erbilgin ◽

Khusan Khodzhaev ◽

Merve Sarıtaş ◽

Ceyhun Bozkurt ◽

...

Keyword(s):

Ligand Binding ◽

B Cell ◽

Candidate Gene ◽

Hodgkin Lymphoma ◽

Genetic Predisposition ◽

Index Case ◽

B Cell Lymphoma ◽

Consanguineous Marriages ◽

Therapeutic Benefits ◽

The Family

Abstract Objective Hodgkin lymphoma (HL) is a lymphoproliferative malignancy of B-cell origin and one of the common tumors in adolescent and young adults. Less than one percent of HL patients have a family history and siblings of the affected individual may have an increased risk of developing the disease. HL risk is associated with immunodeficiency syndromes. Clustering of cases of HL within families may also suggest a genetic predisposition. In cases with familial clustering, recognition of genetic predisposition genes in the pathogenesis of HL may offer therapeutic benefits, and provide genetic counseling and surveillance for the family members for early diagnosis. We had previously reported CD70 signaling defect in an extended family with multiple consanguineous marriages and individuals affected with Hodgkin lymphoma and/or immune deficiency (J Clin Immunol.2020, 40(6):883-892). The aim of this study is to investigate for a cancer predisposition gene/condition in two siblings with HL, whose parents had a consanguineous marriage. Material and Methods A 10 year-old girl with progressive HL was referred for further evaluation and treatment. She had been diagnosed with stage IIIBs HL, nodular sclerosing subtype and had received chemotherapy (ABVD) where she was reported to have partial response, followed by progressive disease. The tumor was CD30 positive. At admission, the patient had a massive enlarged mediastinum due to conglomerate lymph nodes, nodules in the lung, lymph adenomegalies in the cervical, supraclavicular, axillary regions. She was treated with ifosfamide+mesna, etoposide, carboplatinum (ICE) and brentuximab and achieved a complete metabolic response in PET-CT evaluation after six courses. She is transferred to receive stem cell transplantation. Her 24 year-old sister was also diagnosed with early stage HL during pregnancy, had a healthy delivery, was treated with chemotherapy and was in remission. In this study, we performed exome sequencing of the index case (IV-7). The elder sister who was diagnosed with HL and the parents, whose informed consents were taken. There were five children in the family; two of the girls were (IV-7 and IV-11) diagnosed with HL, a boy (IV-8) with inflammatory skin findings, a sister (IV-9) and a cousin (IV-13) with suspected lymphoma, and a healthy girl (IV-10) (Figure 1). After quality control and alignment steps, variants were annotated using ANNOVAR, dbSNP, and ExAC. Variant evaluation was performed by in-house filtering steps based on MAF<0.1%, CADD score >10, SIFT, PolyPhen, MutationTaster, FATHMM scores. We also used Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE) tool for identification and classification of the variants. Results A germline homozygous missense gene variation (c.928 G>A, p.D310N, rs144833620) was identified on the ligand binding domain of the Pregnane X Receptor Gene (PXR or NR1I2) gene. Sanger sequencing confirmed the familial segregation; IV-7 and IV-11 HL cases were homozygous and parents (III-13 and III-14) were heterozygous. The index case did not have elevated EBV-DNA PCR levels. Index case had increased CD3, CD2 and CD8 and reduced CD4 levels. (Conclusion) The PXR encodes a member of the nuclear receptor (NR) superfamily and functions in the metabolism and elimination of xenobiotics and several commonly used drugs. Heterodimerization of PXR and RXR (functional partner) lead to conformational changes upon ligand binding, functions as the transcriptional regulator of PXR target genes including metabolizing enzymes, drug transporters, inflammation and cell cycle associated genes. Previously reported Pxr-/- animal model showed that mice lacking the steroid and xenobiotic receptor develop B cell lymphoma. However, there is no case of HL published linked to variation in PXR gene. Clustering of cases of HL within families, especially in families with consanguineous marriages should suggest a genetic predisposition. The PXR seems to be a novel candidate gene for predisposition of HL. Funding: This study was funded by Scientific Research Projects Coordination Unit of Istanbul University with a project number TDK-2020-37127. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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