4-1BB Monoclonal Antibody Research Articles

Abstract 5558: Discovery of a novel Claudin 6 X 4-1BB bispecific antibody with potent anti-tumor activity through conditional 4-1BB activation

Abstract Background Claudin 6 (CLDN6) is a tight junction transmembrane protein normally only expressed during embryonic development, but is aberrantly expressed in a variety of tumors, including ovarian cancer, testicular cancer, hepatocellular and lung adenocarcinoma. Here, we generated a CLDN6 X 4-1BB bispecific antibody (BsAb) which could conditionally activate T cells through 4-1BB stimulation upon CLDN6 engagement, positioning it as a potential novel immunotherapy for ovarian cancer and other CLDN6 positive tumors. Methods CLDN6 X 4-1BB BsAb was evaluated for its antigen binding through cell-based binding assay. In vitro function of the antibody was confirmed in a 4-1BB signaling reporter assay and primary human PBMC assay. In vivo efficacy of the antibody was further demonstrated in 4-1BB humanized syngeneic mouse model. Ex vivo analysis of tumor infiltrating and peripheral immune cells was conducted by FACS. Safety parameters such as liver enzymes and pathology upon antibody administration were also assessed. Results CLDN6 X 4-1BB BsAb showed strong binding to several ovarian cancer cell lines with different levels of CLDN6 expression. Functional evaluation of CLDN6X4-1BB BsAb indicated that the activation of 4-1BB signaling was dependent on CLDN6 expression on tumor cells and the activity was stronger than reference 4-1BB monoclonal antibody urelumab. In a humanized 4-1BB mouse model, CLDN6X4-1BB BsAb showed tumor growth inhibition superior to the combination of anti-CLDN6 and anti-4-1BB monospecific antibodies, and the treatment effect was associated with an increase in tumor infiltrating CD45 and CD8 cells as well as CD8/Treg ratio. From the safety perspective, there were no significant changes in liver enzymes or liver histopathology following repeated BsAb administration, suggesting little risks for liver toxicity commonly induced by other 4-1BB agonist antibodies. Conclusions We have successfully generated a novel CLDN6-targeted 4-1BB bispecific antibody which could induce potent 4-1BB stimulation and anti-tumor activity in a CLDN6-dependent manner while minimizing the risk of liver toxicity. Taken together, these data support further development of CLDN6 X 4-1BB bispecific antibody towards IND and clinical trial in 2022. Citation Format: Jian Li, Wenqing Jiang, Yan Liu, Zhengyi Wang, Xi Chen, Taylor B. Guo. Discovery of a novel Claudin 6 X 4-1BB bispecific antibody with potent anti-tumor activity through conditional 4-1BB activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5558.

Abstract LB173: APVO603: A dual 4-1BB and OX40 bispecific approach utilizing ADAPTIRTMtechnology designed to deliver a conditional T cell/NK response against solid tumors

Abstract APVO603 is Aptevo's bispecific candidate targeting 4-1BB and OX40. It was designed to have unique properties with the potential to overcome some of the clinical challenges observed with monoclonal antibody targeting these receptors. APVO603 is engineered as an FcγR-signaling deficient bispecific antibody that utilizes Aptevo's ADAPTIR technology for a distinct approach for dual targeting of 4-1BB and OX40 in the absence of additional effector activity. The distinct characteristics of APVO603 may enable conditional activation of 4-1BB and OX40 via agonism of these receptors only when cross-linked via engagement of the other receptor via cis and/or trans cellular interactions. Thus, APVO603 is designed with the potential to overcome both the on-target toxicity and limited efficacy observed with 4-1BB and OX40 monoclonal antibody treatment in the clinic. Anti-4-1BB and OX40 binding domains were optimized to increase affinity, function and stability, then incorporated into the ADAPTIR bispecific antibody platform to produce the APVO603 lead candidate. APVO603 was found to augment 4-1BB and OX40 activity in a dose-dependent manner and is strictly dependent on engagement of the reciprocal receptor to elicit 4-1BB or OX40 signaling in vitro. In preclinical assays using PBMCs sub-optimally stimulated with anti-CD3, APVO603 induces synergistic proliferation of CD4+, CD8+ T and NK cells when compared to anti-OX40 or 4-1BB antibodies with a wt Fc, included either individually or in combination. Additionally, APVO603 enhances proinflammatory cytokine production, granzyme B expression, and reduces the T cell exhaustion phenotype. The mechanistic activity of APVO603 resulted in dose-dependent control of in vivo tumor growth in a preclinical humanized murine xenograft model using established murine MB49 bladder tumors in human 4-1BB and OX40 double knock-in mice. APVO603 is a dual-agonistic bispecific antibody that augments the effector function of activated CD4+ and CD8+ T cells and NK cells in a dose-dependent manner and reduces growth of established tumors in vivo. This preclinical data demonstrates conditional dual stimulation of 4-1BB and OX40 and supports further development of APVO603, a promising immuno-oncology therapeutic with potential for benefit in solid tumors. This program is progressing into IND-enabling studies later this year. Citation Format: Michelle H. Nelson, Robert E. Miller, Secil Franke-Welch, Ruth Chenault, Hang Fang, Allison Chunyk, Gabriela Hernandez-Hoyos, Hilario J. Ramos, David Bienvenue, Catherine McMahan. APVO603: A dual 4-1BB and OX40 bispecific approach utilizing ADAPTIRTMtechnology designed to deliver a conditional T cell/NK response against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB173.

Abstract 5644: Claudin 18.2 X 4-1BB bispecific antibody induced potent tumor inhibition through tumor-specific 4-1BB activation

Abstract Background Claudin 18.2 (CLDN18.2) is a gastric-specific membrane protein. In the healthy tissue, CLDN18.2 expression is restricted to the short-lived differentiated cells of gastric mucosa as a component of tight junction with limited accessibility of antibody treatment. However, it was ectopically expressed at significant levels in a variety of primary lesion and metastases of epithelial tumor entities, including gastric, pancreatic, esophageal, and lung adenocarcinoma cells. Taken together, CLDN18.2 was believed to be an ideal tumor antigen for immunotherapy. Here, we generated and described a bispecific antibody TJ-CD4B which could redirect and activate T cells to CLDN18.2 positive tumor cells by engaging 4-1BB antigen, thus positioned as a potential novel therapy for gastric cancer and other CLDN18.2 positive tumors. Methods TJ-CD4B was evaluated for its antigen binding through protein-based binding and cell based binding assay. The cell-based function of TJ-CD4B was further evaluated in a 4-1BB signaling reporter assay and primary human T cell assay. The in vivo efficacy was validated in 4-1BB humanized C57BL/6 mice transferred with colon cancer cell line MC38 overexpressed with human CLDN18.2. Percentage of tumor infiltrating and peripheral CD8+ T cells was evaluated by FACS analysis. Results TJ-CD4B showed stronger binding capability to CLDN18.2 when compared with benchmark monoclonal antibody against CLDN18.2 (IMAB362). Functional evaluation of TJ-CD4B indicated the activation of 4-1BB signaling was solely dependent on CLDN18.2 expression on the cell. Furthermore, TJ-CD4B showed superior 4-1BB activity than benchmark 4-1BB monoclonal antibody (Urelumab) in the presence of cells expressing a wide range of CLDN18.2 level including CLDN18.2-low cells. Using a humanized 4-1BB mouse model, TJ-CD4B showed strong tumor growth inhibition (TGI) of CLDN18.2 expressing tumor cells. Meanwhile, we also observed TJ-CD4B increased tumor infiltrating lymphocytes while it had no impact on peripheral lymphocytes. Drug developability assessment indicated that TJ-CD4B was a reasonably developable molecule to be taken forward. Conclusions Our data showed that CLDN18.2-targeted 4-1BB bispecific antibody, TJ-CD4B, displayed potent anti-tumor activity only in tumor microenvironment, possibly minimizing the risk of peripheral toxicity. These compelling data supported the further development of TJ-CD4B, which aims to enter clinical phase in early 2021. Citation Format: Wenqing Jiang, Lei Fang, Zhengyi Wang, Taylor B. Guo, Eunyoung Park, Eunsil Sung, Jaeho Jung. Claudin 18.2 X 4-1BB bispecific antibody induced potent tumor inhibition through tumor-specific 4-1BB activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5644.

Study of local application of CpG-oligodeoxynucleotide combined with 4-1BB monoclonal antibody to treat hepatoma- bearing mice

Objective: To investigate the curative effect of local application of CpG-oligodeoxynucleotide (CpG-ODN) combined with 4-1BB monoclonal antibody in hepatoma-bearing mice, and to evaluate the effect of 4-1BB monoclonal antibody on CpG-ODN immunotherapy. Methods: H22 single cell suspension was injected subcutaneously into the axilla and four limbs of the BALB/c male mice to establish a tumor-bearing mice model. After 7 days, 30 mice with corresponding tumor-bearing volume were screened and randomly divided into model control group, CpG group and CpG+4-1BB group, and the drug was injected into the tumors of left lower extremity. The same batch of normal mice was selected as normal control group. Survival of mice was recorded. Tumor-bearing volume and organ index were calculated. Serum levels of interleukin (IL) - 12 and interferon (IFN) gamma and spleen CD8(+)T lymphocyte ratio were measured. The measurement data were analyzed by analysis of variance. The survival rate of each group of mice was analyzed by log-rank test. Results: Mice in the model control group with tumor-bearing volume had a sustained growth before the execution. CpG group and the CpG+4-1BB group [(976.08 ± 29.55) mm(3), (47.25 ± 0.93) mm(3))] tumor-bearing volume was decreased than model group [(1 336.52 ± 39.40) mm3] (F = 5 329.273, P < 0.05). CpG+4-1BB group distant tumor-bearing volume [(611.83 ± 113.02) mm3] was decreased than model group and CpG group [(1 406.62 ± 51.09) mm(3), (1 380.01 ± 51.44) mm3] (F = 247.160, P < 0.05), but there was no significant difference between the CpG group and the model group (P > 0.05). Serum IL-12 concentration (23.90 ± 2.33 pg/ml), IFN-γ concentration (103.02 ± 6.10 pg/ml) and spleen CD8(+)T cell ratio (4.54 ± 0.62%) in the model group were lower than those in the normal group (P < 0.05). Serum IL-12 concentration in CpG group and CpG+4-1BB group (29.21 ± 2.23 pg/ml, 37.04 ± 1.49 pg/ml), IFN-γ concentration (116.12 ± 4.08 pg/ml, 138.65 ± 1.72 pg/ml), CD8(+)T cell ratio (6.65 ± 0.64%, 12.73 ± 0.88%) were higher than the model group, while CpG+4-1BB group was higher than the CpG group (P < 0.05). The survival rate of CpG+4-1BB group was higher than that of model group and CpG group (χ(2) = 25.544, P < 0.05), but there was no significant difference between CpG group and model group (P > 0.05). There was no significant difference in organ index between the four groups (P > 0.05). Conclusion: 4-1BB monoclonal antibody combined with CpG-ODN therapy can shrink hepatoma-bearing capacity, inhibit the growth of distant tumors and significantly prolong the survival time of mice.

IMMU-49. TARGETING CCR2 SIGNALING IN PEDIATRIC MEDULLOBLASTOMA

As chemokines are important mediators of cell–cell communication, we sought to identify commonly expressed chemokines in mouse and human medulloblastoma (MB) lines as surrogates to microglia/MB tumor cell interactions. We found CCL2 (chemokine (C-C motif) ligand 2) messenger RNA to be expressed by medulloblastoma lines. However, these lines did not express the CCL2 receptor, CCR2, which was found on microglia. The MP-1 medulloblastoma cell line is known to have low basal levels of CCL2. Hence, we overexpressed CCL2 plasmid with GFP tagging in the MP-1 medulloblastoma line to investigate the hypothesis that medulloblastoma-secreted CCL2 interacts with microglia to affect medulloblastoma growth and invasion. In medulloblastoma–microglia co-culture models, we observed an increase in the proliferation of CCL2-overexpressing clones. ELISA protein analyses revealed that interleukin-6 (IL-6) was consistently increased in the co-culture. Recombinant IL-6 enhanced the invasion of medulloblastoma cells when these were cultured alone, whereas a neutralizing antibody to IL-6 attenuated the microglia-stimulated invasion. This study has uncovered a mechanism by which medulloblastoma cells exploit microglia (M2-phenotype) for increased invasion. Specifically, medulloblastoma-derived CCL2 acts upon CCR2-bearing tumor-associated microglias (TAMs), which produce IL-6 to stimulate medulloblastoma cells, in a CCL2/CCR2/IL-6 loop. We targeted this loop by combining radiation therapy with 4-1BB (CD-137) monoclonal antibody (mAb), as CD-137 is involved in regulation of immune cell proliferation and survival. We re-educated microglia cells toward a cytotoxic M1-like phenotype and amplified effective anti-tumor immune responses in in vitro co-culture models. ELISA spot shows treatment with 50 ng of 4-1BB (CD137) + 1Gy X-ray radiation converted the microglia from M2 to M1 phenotype via a decrease in CCR2. This treatment induces the M1 phenotype microglia to induce MP-1 cell death. Based on our results, the use of 4-1BB mAb plus low-dose single fraction radiation may prove to be beneficial for the treatment of medulloblastoma.

4-1BB Aptamer-Based Immunomodulation Enhances the Therapeutic Index of Radiation Therapy in Murine Tumor Models

Toreport a novel strategy using oligonucleotide aptamers to 4-1BB as an alternate method for costimulation, and show that combinatorial therapy with radiation improves the therapeutic ratio over equivalent monoclonal antibodies. Subcutaneous 4T1 (mouse mammary carcinoma) tumors were established (approximately 100mm(3)), and a radiation therapy (RT) dose/fractionation schedule that optimally synergizes with 4-1BB monoclonal antibody (mAb) was identified. Comparable tumor control and animal survival was observed when either 4-1BB antibody or aptamer were combined with RT using models of breast cancer and melanoma (4T1 and B16-F10). Off-target CD8(+) T-cell toxicity was evaluated by quantification of CD8(+) Tcells in livers and spleens of treated animals. When combined with 4-1BB mAb, significant differences in tumor control were observed by varying RT dose and fractionation schedules. Optimal synergy between RT and 4-1BB mAb was observed at 5Gy×6. Testing 4-1BB mAb and aptamer independently using the optimal RT (5Gy×6 for 4T1/Balb/c and 12Gy×1 for B16/C57BL6J mouse models) revealed equivalent tumor control using 4-1BB aptamer and 4-1BB mAb. 4-1BB mAb, but not 4-1BB aptamer-treated animals, exhibited increased lymphocytic liver infiltrates and increased splenic and liver CD8(+) Tcells. Radiation therapy synergizes with 4-1BB mAb, and this effect is dependent on RT dose and fractionation. Tumor control by 4-1BB aptamer is equivalent to 4-1BB mAb when combined with optimal RT dose, without eliciting off-target liver and spleen CD8(+) expansion. 4-1BB aptamer-based costimulation affords a comparable and less toxic strategy to augment RT-mediated tumor control.

Differential effects of agonistic 4-1BB (CD137) monoclonal antibody on the maturation and functions of hypoxic dendritic cells.

Agonistic 4-1BB monoclonal antibody (mAb), a promising approach for tumor immunotherapy, has entered clinical trials for some tumors due to its immunostimulatory effects on immune cells. Hypoxia, the hallmark of tumor microenvironment, influences functions of immune cells including dendritic cells (DCs). It remains unestablished whether 4-1BB mAb takes effects on DCs in hypoxic microenvironment. This study aims to examine the role of agonistic 4-1BB mAb in the maturation and functions of murine hypoxic DCs. As expected, hypoxia suppressed the maturation and activation of DCs, as suggested by down-regulation of class II MHC, co-stimulatory molecules and proinflammatory cytokines. These inhibitory effects of hypoxia were partially reversed by triggering 4-1BB on DCs with agonistic mAb, as evidenced by elevated co-stimulatory molecules CD80, CD86, and proinflammatory cytokines such as IL-6, TNF-α. Unexpectedly, the ability of hypoxic DCs to stimulate CD4+T cell proliferation seemed not to be affected by agonistic 4-1BB mAb. These data demonstrate that agonistic 4-1BB mAb partially restores the phenotypic maturation and proinflammatory function of hypoxic DCs, but fails to rescue their ability to stimulate T cell response. Collectively, our study provides evidence on the efficiency of agonistic 4-1BB mAb in hypoxic microenvironment, deserving of further consideration for clinic application.

Treatment with Anti-CD137 (41BB) mAb enhances peptide vaccination against respiratory syncytial virus in aged mice (106.2)

Abstract Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness (LRI) in infants and also a major cause of morbidity and mortality in the elderly. Using an aged mouse model of RSV pathogenesis, we found that aged mice had impaired antigen-specific CD8 T cell responses and delayed RSV clearance compared to young mice. We generated a peptide vaccine approach, TriVax, which is a co-mixture of a peptide representing an immunodominant RSV CD8 T cell epitope, a Toll-like receptor agonist, and a costimulatory anti-CD40 antibody. TriVax vaccination generated robust CD8 T cell responses and had a protective effect against RSV challenge in young BALB/c mice but not in aged BALB/c mice. We hypothesized that treatment of aged mice with agonistic CD137 (41BB) monoclonal antibody would stimulate T cells and enhance TriVax efficacy to RSV challenge in aged mice. We immunized 18-month old BALB/c mice twice with TriVax + anti-CD137 mAb. We found that co-administration of anti-CD137 mAb with TriVax enhanced antigen-specific CD8 T cell responses as well as showed a protective effect after RSV challenge. Thus, anti-CD137 mAb treatment rescued the deficient response to RSV TriVax vaccination in aged mice. The 41BB co-stimulatory pathway may be a target for enhancing T cell responses in the elderly.

Investigation of the Effective Dose of Agonistic 4-1BB Monoclonal Antibody in a Murine Colon Cancer Metastasis Model

Purpose: The aim of this study was to find the dose of agonistic 4-1BB monoclonal antibody (㎃b) that results in optimal T cell activation. Methods: Cancer was induced in mice by an intrahepatic parenchymal injection of 1×10? cells of CT26 cells. Cancer-carrying mice (n=84) were divided into seven groups and treated with either rat IgG or agonistic 4-1BB monoclonal antibody (㎃b) (5㎍, 10㎍, 20㎍, 100㎍, 200㎍, or 300㎍). All treatments were administered intraperitoneally on days 7, 9, and 11. Mice from each group were sacrificed on days 14, 28, and 42. Harvested livers were weighed and the numbers of T cells in the splenocytes were analyzed with a FACS Vantage flow cytometer. Results: Liver weights increased when 5㎍ of agonistic 4-1BB ㎃b was administered, but showed no additional weight increase for doses greater than 10㎍. The absolute numbers of CD4＋ and CD8＋ T cells increased in groups treated with low doses of agonistic 4-1BB ㎃b (5㎍, 10㎍, or 20㎍), but did not increase in the groups treated with high doses of ㎃b (100㎍, 200㎍, or 300㎍). The levels of CD4/annexin V and CD8/annexin V increased as the dose increased, and the absolute cell numbers of CD4/annexin V were greater than those of CD8/annexin V. Conclusion: Liver weight, including the cancer mass, failed to increase at agonistic 4-1BB ㎃b doses greater than 10㎍. A high dose (≥100㎍) of agonistic 4-1BB ㎃b resulted in lower counts of absolute T cells. This study suggests that a low dose (20㎍) of agonistic 4-1BB ㎃b can be used for optimal T cell activation in combination with other anti-cancer treatments.

Location of extracellular cysteine-rich domains of 4-1BB binding to murine 4-1BB ligand and analysis of its possible structure

ObJective To locate the cysteine-rich domains(CRD) of murine 4-1BB binding to its natural ligand. Methods A serial soluble extracellular CRDs of routine 4-1BB and 4-1BBL fusion proteins was constructed and prepared. The binding of purified 4-1BB-Igs to 4-1BBL and 4-1BB monoclonal antibody were tested using ELISA assay and Western blot analysis. Blocking experiment with 4-1BBL and 4-1BB mon-oclonal antibody was performed by ELISA assay. Results All truncated overlapped proteins containing ex-tracellular CRD Ⅱ of murine 4-1BB were able to bind to 4-1BBL by ELISA assay, excepting the CRD Ⅰ do-main alone. A 4-1BB monoclonal antibody proved to block the interaction of 4-1BB and 4-1BBI, was also able to bind to CRD Ⅱ. Conclusion Murine 4-1BBL whose specificity was mapped to CRD Ⅱ of 4-1BB ex-tracellular region with a possible conformational structure. Key words: Murine 4-1 BB molecule; Cysteine-rich domain; Recognition region of 4-1BBL

Augmentation of SIV DNA vaccine-induced cellular immunity by targeting the 4-1BB costimulatory molecule

DNA vaccines are effective at inducing antigen-specific cellular immune responses. Approaches to improve these responses, however, are needed. We examined the effect of stimulating 4-1BB, an activation-inducible T-cell costimulatory receptor, by intravenously co-administering anti-human 4-1BB monoclonal antibody (mAb) in DNA-immunized cynomolgus macaques. Three groups of six cynomolgus macaques were immunized intramuscularly with a DNA vaccine encoding SIV Gag antigen (pSIVgag) at weeks 0, 4 and 8. At days 12, 15, and 19, six macaques received anti-4-1BB 4E9 mAb and six macaques received anti-4-1BB 10C7 mAb. Treatment with 10C7 mAb led to a significant augmentation of SIV Gag-specific IFN-gamma, granzyme B and perforin responses. Treatment with humanized 4E9 mAb also resulted in an enhancement of SIV Gag-specific cellular responses but the magnitude was lower compared to animals receiving 10C7 mAb. These responses persisted up to week 40 and were mostly mediated by CD8(+) T cells. Treatment with anti-4-1BB mAb was more effective in driving the CD8(+) T cells toward a more differentiated CCR7(-)/CD45RA(+) effector state. This study demonstrates that targeting the 4-1BB molecule in vivo results in an enhanced and long-lasting cellular immune response. 4-1BB stimulation may be a promising approach to enhance the effectiveness of DNA vaccines.

Immunotherapy Targeting 4-1BB and Its Ligand

T-cell activation in the absence of costimulation is futile because T-cells deprived of costimulatory signals enter a state of unresponsiveness or anergy. The interaction of 4-1BB and 4-1BB ligand (4-1BBL) activates an important costimulatory pathway with diverse and important roles in immune regulation. Signals relayed through 4-1BB generate strong CD8(+) T-cell responses rather than CD4(+) T-cell responses; this action results in cytokine induction and promotes T-cell survival. In recent years, 4-1BB-mediated immune regulation has gained great significance because of the seemingly contradictory dual roles of agonistic anti-4-1BB in vivo disease models. To date, agonistic 4-1BB monoclonal antibody has shown therapeutic potential against a variety of tumors, CD4(+) T-cell-mediated autoimmune diseases, and chronic graft-versus-host disease. In addition, blockade of 4-1BB/4-1BBL interaction has produced therapeutic effects against coxsackievirus-induced myocardial inflammation, herpetic stromal keratitis, and graft rejection. We propose that the dual roles of agonistic anti-4-1BB--an enhanced effector function and a suppressor function--are mediated by a novel CD11c(+)CD8(+) T-cell population.

Stimulation with 4-1BB (CD137) inhibits chronic graft-versus-host disease by inducing activation-induced cell death of donor CD4+ T cells

Abstract4-1BB, a member of the tumor necrosis factor (TNF) receptor superfamily, is a costimulator for activated T cells. Previous studies have established that treatment with agonistic anti–4-BB monoclonal antibody (3H3) is effective in reversing the progression of spontaneous systemic lupus erythematosus. Its therapeutic effect is mediated by suppression of autoantibody production. In this report, we show that a single injection of 3H3 blocks chronic graft-versus-host disease (cGVHD) in the parent-into-F1 model. In particular, donor CD4+ T cells are rapidly eliminated from host spleens by activation-induced cell death after 4-1BB triggering. Since donor CD4+ T cells are required for the development of cGVHD, and 3H3-mediated inhibition of autoantibody production occurs without donor CD8+ T cells, 3H3 blocks cGVHD by preventing alloreactive donor CD4+ T cells from activating host B cells. Importantly, 3H3 treatment can reverse the progression of advanced cGVHD. Our findings indicate that agonistic anti–4-1BB monoclonal antibody has potential as an immunotherapeutic agent for preventing and treating cGVHD.

Humanization of agonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library

Given the key role 4-1BB plays in the stimulation of T cells, humanization of agonistic anti-human 4-1BB monoclonal antibody (mAb) may have important clinical applications. In this paper, we present the humanization of agonistic anti-human 4-1BB mAb, BBK-4, using a phage display library. We first prepared the combinatorial library by incorporating murine and human alternative at positions representing buried residues that might affect the structural integrity of the antigen binding site. Six humanized single chain Fv (scFv) fragments were selected from the combinatorial library expressing phage-displayed humanized scFv. They were found to retain the epitope specificity of the original mAb but had affinities of lower than 1/10 of the original. In spite of the lower affinity, the humanized scFv coated on the surface expanded human peripheral blood mononuclear cells (PBMCs) in MLR similarly to the original mAb in the presence of anti-CD3 mAb. These results suggest that humanized anti-human 4-1BB scFvs can be used as a valuable reagent for clinical application.

Improving efficacy of interleukin-12-transfected dendritic cells injected into murine colon cancer with anti-CD137 monoclonal antibodies and alloantigens.

Intralesional administration of cultured dendritic cells (DCs) engineered to produce IL-12 by in vitro infection with recombinant adenovirus frequently displays eradicating efficacy against established subcutaneous tumors derived from the CT26 murine colon carcinoma cell line. The elicited response is mainly mediated by cytolytic T lymphocytes. In order to search for strategies that would enhance the efficacy of the therapeutic procedure against less immunogenic tumors, we moved onto malignancies derived from the inoculation of MC38 colon cancer cells that are less prone to undergo complete regression upon a single intratumoral injection of IL-12-secreting DCs. In this model, we found that repeated injections of such DCs, as opposed to a single injection, achieved better efficacy against both the injected and a distantly implanted tumor; that the use of semiallogeneic DCs that are mismatched in one MHC haplotype with the tumor host showed slightly better efficacy; and that the combination of this treatment with systemic injections of immunostimulatory anti-CD137 (4-1BB) monoclonal antibody achieved potent combined effects that correlated with the antitumor immune response measured in IFN-gamma ELISPOT assays. The elicited systemic immune response eradicates concomitant untreated lesions in most cases. Curative efficacy was also found against some tumors established for 2 weeks when these strategies were used in combination. These are preclinical pieces of evidence to be considered in order to enhance the therapeutic benefit of a strategy that is currently being tested in clinical trials. Supplementary Material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

Humanization of antagonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library.

Anti-4-1BB (CD137) monoclonal antibody (mAb) has been reported to suppress immune responses and to have the potential for use as a therapeutic agent to block autoimmune diseases. Previously, the authors prepared an antagonistic anti-human 4-1BB (CD137) mAb, BBK2. Here the authors report the humanization of BBK2 using a phage display library. Four humanized single-chain Fv (scFv) fragments were selected from a combinatorial library expressing a phage-displayed humanized scFv. They were found to retain the epitope specificity of the original mAb and to have affinities higher than those of the original. Both the soluble and bound forms of the humanized scFv suppressed the proliferation of human peripheral blood mononuclear cells, similar to the original mAb. These results suggest that humanized anti-human 4-1BB scFvs can be used as a valuable reagent for clinical application.

Characterization of Immunological and Molecular Properties of an Anti-human 4-1BB Monoclonal Antibody

Characterization of Immunological and Molecular Properties of an Anti-human 4-1BB Monoclonal Antibody