ALBERT

Description: B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-α, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis.

Description: Immunotherapy using anti-tumor antibodies has become a feasible alternative for treating patients with lymphoma. These anti-tumor antibodies may target a specific receptor to disrupt proliferative signaling or mediate their anti-tumor effect by antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated killing. The CD40 antigen is a good target for such anti-tumor antibodies for several reasons: CD40 is expressed on the vast majority of the non-Hodgkin’s B cell lymphomas and it has been proposed that the CD40/CD40L interaction provides a critical survival or proliferative signal for B cell lymphoma, especially the low-grade follicular lymphoma. In addition, B lymphoma cell lines become less sensitive to chemotherapy-induced apoptosis after CD40 cross-linking in an in vitro study. Therefore, an anti-CD40 antagonist that disrupts the CD40/CD40L interaction and mediates effector mechanism could have a therapeutic advantage. In this report, we describe a fully human anti-CD40 antagonistic IgG1 monoclonal antibody, CHIR-12.12 that was generated from mice with a human immunoglobulin gene loci (XenoMouse®mice, Abgenix Inc.). We first compared the antigen expression level of CD40 to the level of CD20, the target for rituximab, on primary lymphoma cells. While the expression level of CD40 was similar between different samples of primary follicular lymphoma cells, it was 10 fold less than the level of CD20. The expression of CD40 and CD20 on chronic lymphocytic leukemia/small lymphocytic lymphoma cells (CLL/SLL) was more variable. However, the level of CD20 was still significantly higher than the level of CD40 in all samples tested (2.4 to 13 fold). While CHIR-12.12 binds to primary lymphoma cells similarly to several other anti-CD40 antibodies, CHIR-12.12 did not induce proliferation of these primary tumore cells. By contrast, an agonist anti-CD40 antibody induced proliferation of these lymphoma cells up to 6-fold over baseline. To study the ability of CHIR-12.12 to interrupt the CD40-CD40L interaction, we cultured lymphoma cells with CD40L-transfected feeder cells in the presence of control IgG1, CHIR-12.12 or rituximab. In this system, the lymphoma cells proliferate in response to CD40-CD40L interaction. The addition of rituximab did not influence the proliferation. However, CHIR-12.12 inhibited the proliferation of follicular lymphoma and of CLL/SLL cells in a dose-dependent manner. The inhibition was observed with antibody concentration at 1 μg/ml and reached maximum of 90% inhibition at 10 μg/ml. We then evaluated the ability of CHIR-12.12 to elicit complement-mediated killing or ADCC. In vitro, rituximab induced complement-mediated cytotoxicity, while CHIR-12.12 did not. However, both CHIR-12.12 and rituximab induced effective ADCC of primary follicular lymphoma cells using purified NK cells from a healthy donor. Even though the level of CD40 is 10-fold less than the level of CD20 on the cell surface of these tumor cells, CHIR-12.12 induced the same degree of ADCC killing as did rituximab. Thus, this novel antagonist CHIR-12.12 antibody both blocks tumor-stimulatory CD40/CD40L interaction and mediates ADCC in the presence of a low number of target antigen. Our results support further development of this antibody to treat patients with B cell lymphoma.

Description: Rituximab is a chimeric monoclonal antibody that targets B-cell–specific antigen CD20 and an effective treatment for B-cell non-Hodgkin lymphoma. Although it is readily used in clinical practice, the exact mechanism of its antitumor effect is unclear. One potential mechanism involves complement-mediated cytotoxicity. It has been shown that rituximab induces complement-mediated cytotoxicity in follicular lymphoma cells in vitro, and complement inhibitors CD55 and CD59 may regulate this process. To determine whether complement inhibitors play a role in regulating the antitumor effect of rituximab, the expression of complement inhibitors CD46, CD55, and CD59 was analyzed in pretreatment tumor cells from 29 rituximab-treated follicular lymphoma patients. Among them, 8 patients achieved complete responses, 11 patients achieved partial responses, and 10 patients showed no or minimal responses to rituximab treatment. Expression of surface CD20, CD46, CD55, and CD59 was determined by 2-color flow cytometry. Although the CD59 level was slightly lower in the complete response group, there was no statistically significant difference in the expression of individual complement inhibitor CD46 (mean channel fluorescence [MCF]: NR, 26.4; PR, 21.9; CR, 29.9), CD55 (MCF: NR, 16.4; PR, 14.9; CR, 23.2), or CD59 (MCF: NR, 41.6; PR, 40.6; CR, 30.6), the combination of any 2 inhibitors, or all 3 on tumor cells from 3 response groups. In addition, there was no difference in the rituximab-induced complement-mediated cytotoxicity in an in vitro assay using tumor cells from 3 response groups. Thus, CD46, CD55, and CD59 expression on pretreatment tumor cells, or their susceptibility to in vitro complement-mediated killing, does not predict clinical outcome after rituximab treatment.

Description: A hematopoietic cell transplantation regimen was adapted from a preclinical model that used reduced-intensity conditioning (RIC) and protected against graft-versus-host disease (GVHD) by skewing residual host T-cell subsets to favor regulatory natural killer T cells. One hundred eleven patients with lymphoid (64) and myeloid (47) malignancies received RIC using total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) followed by the infusion of granulocyte colony-stimulating factor-mobilized grafts. Included were 34 patients at least 60 years of age, 32 patients at high risk of lymphoma relapse after disease recurrence following prior autologous transplantation, and 51 patients at high risk of developing GVHD due to lack of a fully human leukocyte antigen (HLA)–matched related donor. Durable chimerism was achieved in 97% of patients. Cumulative probabilities of acute GVHD (grades II-IV) were 2 and 10% of patients receiving related and unrelated donor grafts. Nonrelapse mortality (NRM) at 1 year was less than 4%. Cumulative incidence of chronic GVHD was 27%. The 36-month probability of overall and event-free survival was 60% and 40%, respectively. Disease status at start of conditioning and the level of chimerism achieved after transplantation significantly impacted clinical outcome. The high incidence of sustained remission among patients with active disease at time of transplantation suggests retained graft-versus-tumor reactions. Active trial registration currently at clinicaltrials.gov under IDs of NCT00185640 and NCT00186615.

Description: There is indirect but intriguing evidence that complement plays a role in the clinical response to rituximab and other mAb-based therapies of cancer. We identified a non-coding polymorphism in the C1qA component of complement that appears to result in a post-translational splice variant of the C1qA protein. We examined this polymorphism in 90 patients with follicular lymphoma who were treated with single agent rituximab to assess whether a correlation exists with clinical efficacy. The presence of an A versus a G allele at the C1qA[276] locus was determined using restriction fragment length polymorphism analysis by investigators blinded to the clinical outcome of the patients. The molecular and clinical data was then analyzed according to C1qA polymorphism, including measurement of radiographic response and duration of response, using methods similar to those used to evaluate the correlation between polymorphisms in CD16/CD32 genes and clinical outcome. No statistically significant difference in response rate was found based on C1qA polymorphism. However, prolonged remission was noted among those subjects that achieved remission (either PR or CR) for individuals who were carriers of the A allele at the C1qA[276] locus (AA or AG) compared to homozygous GG subjects. The strongest correlation was found among those subjects that achieved a complete remission to single agent rituximab. In this group, GG subjects had a time to progression of 250 days, while AA /AG subjects had time to progression of 830 days (HR=4.1, 95% CI=2.1–96.9). Multivariate Cox regression analysis of the joint effects of the C1qA, CD32 and CD16 polymorphisms showed that C1q[276] A allele was an independent factor for good prognosis, while FcgRIIIa 158 phenylalanine carrier and FcgRIIa 131 arginine carrier indicated poor survival. Thus, the relative risk was 0.2 for C1qA[276] AA/AG versus GG after controlling for CD32 and CD16 polymorphisms. The relative risks were 4.8 for CD32 HR versus HH and 6.16 for CD32 RR versus HH after controlling for complement and CD16 polymorphisms. The relative risks were 4.57 for CD16 VF against VV and 3.69 for CD16 FF versus VV after controlling for complement and CD32 polymorphisms. These data suggest polymorphisms in C1qA may impact on duration of response to rituximab therapy of follicular lymphoma. Ongoing studies are expanding this cohort, assessing whether this polymorphism correlates clinically with outcome in other malignancies or with other therapeutic approaches in lymphoma patients, and exploring the functional significance of this C1qA polymorphism. If further studies confirm that C1qA genetic polymorphisms correlate with duration of response to rituximab, it will have major implications on our understanding of the role of complement in the immune response to lymphoma, and on development of the next generation of mAb-based cancer treatments. Total AA+AG GG p (log-rank test) TTP=Median time to progression (days) All Subjects 90 70 20 Responders 59 46 13 CR 27 22 5 TTP Overall 177 173 191 0.64 TTP Responders 352 456 334 0.12 TTP CR 798 830 250 0.007 Complement C1qA polymorphism associates with freedom from progression in follicular lymphoma patients who achieve a CR after anti-CD20 therapy. Progression-free survival logrank curves were plotted by C1qA[276] AA and AG vs GG genotype. Cheson criteria were used to estimate complete clinical remission after one round of anti-CD20 therapy. Complement C1qA polymorphism associates with freedom from progression in follicular lymphoma patients who achieve a CR after anti-CD20 therapy. Progression-free survival logrank curves were plotted by C1qA[276] AA and AG vs GG genotype. Cheson criteria were used to estimate complete clinical remission after one round of anti-CD20 therapy.

Description: Background: High dose chemotherapy and autologous hematopoietic cell transplantation (AHCT) is the most effective treatment for recurrent and refractory Hodgkin lymphoma (HL). Disease recurrence is still a major cause of treatment failure. Augmented BCNU-regimens are reported to have good outcomes but greater pulmonary toxicity. A novel transplant regimen incorporating gemcitabine and vinorelbine, active drugs in HL different from alkylating agents, was tested to establish the maximum tolerated dose (MTD) of gemcitabine and to assess safety and efficacy at the MTD. Methods: In this phase I/II study, dose escalation was performed with gemcitabine in combination with vinorelbine followed by BCNU at reduced dose, etoposide (VP-16), cyclophosphamide (CY) and AHCT. The first 7 patients had dose escalation of gemcitabine before determining the MTD at 1250 mg/m2 on the basis of elevated liver transaminases and a symptom complex of fever, headache, and skin toxicity. For phase II, a total of 92 patients with recurrent or refractory HL have been treated at the MTD to establish safety and efficacy. The regimen consists of gemcitabine 1250 mg/m2 IV at 10 mg/min and vinorelbine 30 mg/m2 on day-13 and day-8, BCNU 10 mg/kg on day-6, VP-16 60 mg/kg on day-4, CY 100 mg/kg on day-2. Regimen-related toxicity, freedom from progression (FFP) and overall survival (OS) were endpoints of the trial. Results: 70 patients were high risk (based on stage IV disease at relapse, failure to achieve minimal disease, or B symptoms at relapse) and 22 patients were low risk (having no risk features). Median age was 33 years. Median follow-up is 2 years (.26–6.8 years). Median time to neutrophil engraftment was 10 days. Regimen-related toxicities of grade 3 skin rash, fever, headache and liver transaminase elevations were transient. Remarkable was the reduction in incidence of systemic steroid-requiring pulmonary toxicity

Description: Introduction MCL has a poor prognosis. In eligible patients, intensifying frontline, CHOP-like regimens (e.g., cytarabine) as well as high-dose chemotherapy and autologous stem cell transplant (HDT/ASCT) consolidation in first remission have improved progression free survival (PFS) but less so, overall (OS). Preclinical animal models show benefits of adding tumor-specific T-cells to ASCT. CpG (PF-3512676) is a toll-like receptor 9 (TLR9) agonist and an effective vaccine adjuvant that induces costimulatory molecule expression on both antigen-presenting and MCL cells. This phase I/II clinical trial (NCT00490529) adds autologous T-cell transfer, harvested from patients after vaccination with CpG-activated autologous MCL cells - a maneuver termed immunotransplant. This is a planned interim analysis for safety and efficacy triggered by the first 20 patients reaching 1 year post ASCT. Methods Prior to therapy, subjects' tumor cells are collected by biopsy or apheresis and patient-specific vaccine is created by incubating fresh tumor cells with CpG (3 mcg/mL PF-3512676, 72-hr culture), then irradiated and cryopreserved. Patients receive induction chemoimmunotherapy of physician's choice. Patients achieving at least partial response (PR) then receive 3 subcutaneous autologous tumor vaccinations (1 x 108 cells/dose) mixed with CpG (18 mg) every 4-7 days. Primed T-cells (≥ 1 x 1010 CD3 cells) were collected by apheresis 2-4 weeks following vaccine 3 and a rituximab (375 mg/m2) B-cell purge. After standard HDT/ASCT (conditioning = BCNU, cyclophosphamide, etoposide), primed T-cells and a 4th vaccination are given on day+1. A 5th CpG-MCL vaccination followed 3 months post ASCT. The primary endpoint of this study is freedom from minimal residual disease (MRD) at 1 year post ASCT, measured by presence of patient-specific, malignant B-cell VDJ sequence in peripheral blood (ClonoSeq™, analyzed at a sensitivity of ≥ 1 clone/10,000 leukocytes) -an endpoint previously shown to be highly prognostic. Secondary endpoints include PFS, OS, and immune response to vaccine. 59, transplant-eligible, MCL patients are targeted for accrual in this 2-stage design. Results In this interim analysis of 24 patients accrued, 20 have surpassed 1 year post ASCT. All patients had Stage IV disease. Median values included (range): follow-up 43.5 months (11.5-60.1), age 60 years (47-70), and MIPI score 5.9 (5.1-7.8). Vaccine was made from biopsy alone (n=12), apheresis alone (n=9), or both (n=1). Frontline therapy included R-CHOP (n=7), R-hyperCVAD (n=14), alternating R-CHOP/R-DHAP (n=2), and R-EPOCH (n=1). 19 patients achieved complete response while another 3 had PR. All responding patients were vaccinated, able to yield sufficient T-cells for adoptive transfer, and proceeded through standard HDT/ASCT. At 1-year post ASCT, freedom from MRD was 90.5% (n=21). 2 patients did not reach 1-year post ASCT. One died from an ASCT complication (metapneumovirus) while the other relapsed 6 months following ASCT (included in MRD analysis). The most common toxicity due to CpG-MCL vaccine was erythematous rash at injection site (90.9%, n=20, each grade 1). No serious adverse events were seen related to vaccines or adoptive T-cell transfer. All patients had successful hematopoietic recovery, but two were delayed and received backup stem cell infusions with eventual recovery. Though median PFS and OS have not been reached, 3-year PFS and OS at interim analysis are 54.5% and 63.6%, respectively (intention to treat). In this data set, higher expression of co-stimulatory molecules (such as CD40, CD80, and CD86) on a subject's MCL in response to CpG was associated with freedom from MRD (p =0.02). Post-ASCT, higher peripheral T-cell granzyme and perforin response to ex vivo re-challenge with autologous MCL cells was also associated with freedom from MRD (p =0.01). Conclusions The addition of CpG-activated, whole MCL vaccination and autologous, adoptive T-cell transfer to standard therapy appears both feasible and safe. At interim analysis, the 1-year freedom from MRD surpasses rates previously reported in other studies and warrants further investigation. To date, 46 patients have either completed and/or are continuing to undergo study treatment and the study remains open to accrual for patients newly diagnosed with MCL. Disclosures Miklos: Pharmacyclics: Research Funding. Rezvani:Pharmacyclics: Research Funding. Faham:Adaptive Biotechnologies: Employment. Levy:Immune Design: Research Funding; Dynavax: Research Funding.

Description: Multiple treatment modalities are available for MF, but most result in inevitable relapse. Therefore, new treatment strategies that improve response rate and prolong response duration are greatly needed. TSEBT is a highly effective therapy in MF. LD-TSEBT (12 Gy) is much more tolerable than the conventional 36+ Gy dose, thereby allowing for re-treatment; however, LD-TSEBT has a less favorable complete response rate and response duration. Combining LD-TSEBT with immunostimulatory modalities in MF has a strong biological rationale, since radiation-induced exposure of cancer-specific antigens should be synergistic with concomitant stimulation of anti-cancer immune responses. Interleukin-12 is a robust candidate for radioimmunotherapy, as IL-12 has significant anti-MF activity as monotherapy, is very well tolerated without overlapping toxicity with TSEBT, and is a potent stimulator of innate and adaptive immunity. We report on a single-arm open-label phase 2a trial of combination of LD-TSEBT and NM-IL-12. Ten patients are planned for enrollment. Eligibility includes MF-type CTCL stages IB-IIIB and patients must be eligible for LD-TSEBT. TSEBT is started on study day 1 (fractionated 4 Gy/week, up to 12 Gy). NM-IL-12 is administered subcutaneously at 150 ng/kg on days 2 and 15, followed by 6 maintenance doses q4w at 100 ng/kg. The primary endpoint is safety with secondary endpoints being response rate and PFS. Currently, 6 patients are enrolled, 5 evaluable for response; 4 male; median age 55; three have stage IB, one IIB and one IIIB. Median number of previous therapies is 2 (0-6). The treatment was well-tolerated with only grade 1 or 2 AEs; most common AEs include grade 1 headache and chills. One patient achieved CR, 2 PR, and 2 SD. Median follow-up is so far 15 weeks and 5 patients remain on study. One patient has been withdrawn from the study due to development of a suspected PLC (pityriasis lichenoides chronica)-like skin reaction requiring topical steroid therapy. PK and PD analysis was completed in the first 4 patients. It demonstrated measurable drug levels in all patients studied, Cmax being 10.8-56.1 pg/ml achieved at 5-24 hours post injection. Interferon-γ and IP-10 (hallmark PD markers of IL-12 activity) were measurable after the first and the second injections in all patients. Levels of the inhibitory cytokine IL-10 were generally measurable after NM-IL-12 injections, but were very low (