Vaccination with anti-idiotype antibody ganglidiomab mediates a GD2-specific anti-neuroblastoma immune response

Purpose

Immunotherapy targeting disialoganglioside GD₂ emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a GD₂-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD₂ antibodies of the 14.18 family.

Experimental design and results

Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG₁). This antibody bound to anti-GD₂ antibodies 14G2a, ch14.18/CHO, hu14.18, and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD₂ antibodies to the nominal antigen GD₂ as well as GD₂-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD₂ surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD₂ antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD₂-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD₂-specific killing of neuroblastoma cells.

Conclusion

We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.     

Functional Bioassays for Immune Monitoring of High-Risk Neuroblastoma Patients Treated with ch14.18/CHO Anti-GD2 Antibody

Effective treatment of high-risk neuroblastoma (NB) remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD₂ antibody (mAb) ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO) cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD₂-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based bioassays for complement-dependent- (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were set up based on patient serum and immune cells tested against NB cells. For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T) cell ratio of 20∶1 for PBMC preparations as best suited for GD₂-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 40∶1. Optimal results for CDC were found with a serum dilution at 1∶8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV) were below 20%. Sample quality following storage at room temperature (RT) showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m²) revealed GD₂-specific increases in CDC (4.5–9.4 fold) and ADCC (4.6–6.0 fold) on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis.     

Enhancement of cytotoxic and proliferative responses of lymphocytes from melanoma patients by incubation with monoclonal antibodies against ganglioside GD3

Summary Previous studies have shown that monoclonal antibodies (M.Ab) to the ganglioside GD₃ may induce partial remissions in tumour growth in patients with melanoma. In vitro studies demonstrated that M.Abs to GD₃ may also enhance lymphocyte responses to phytohemagglutinin and interleukin 2 (IL2). The present study extended these findings by showing that the IL2-dependent proliferative and cytotoxic response of T cell clones derived from a melanoma patient and a patient with the Vogt-Koyanagi-Harada syndrome were enhanced by pre-incubation of T cells with M.Ab to GD₃. The degree of enhancement increased with the duration of pre-incubation from 2 to 24 h and applied to both T4⁺ and T8⁺ clones. The potentiation of these responses was not specific for M.Abs to GD₃ but was also seen with M.Abs to GD₂ and the T10 structure on T cells but not with M.Abs to the transferrin receptor or an isotype control M.Ab. Incubation of lymphocytes from a melanoma patient with M.Ab to GD₃ during culture with autologous melanoma cells enhanced the proliferative response to the tumour. The expression of IL2 receptors (Tac epitope) on the T cells showed variable enhancement after incubation with M.Ab to GD₃ but the significance of these findings in relation to the mechanism of the enhanced responses is not known. These results suggest that certain M.Abs may stimulate cell-mediated immune responses against tumour cells and that this may provide an additional mode of action of M.Abs against tumours in vivo     

Improved in vivo anti-tumor effects of IgA-Her2 antibodies through half-life extension and serum exposure enhancement by FcRn targeting

Antibody therapy is a validated treatment approach for several malignancies. All currently clinically applied therapeutic antibodies (Abs) are of the IgG isotype. However, not all patients respond to this therapy and relapses can occur. IgA represents an alternative isotype for antibody therapy that engages FcαRI expressing myeloid effector cells, such as neutrophils and monocytes. IgA Abs have been shown to effectively kill tumor cells both in vitro and in vivo. However, due to the short half-life of IgA Abs in mice, daily injections are required to reach an effect comparable to IgG Abs. The relatively long half-life of IgG Abs and serum albumin arises from their capability of interacting with the neonatal Fc receptor (FcRn). As IgA Abs lack a binding site for FcRn, we generated IgA Abs with the variable regions of the Her2-specific Ab trastuzumab and attached an albumin-binding domain (ABD) to the heavy or light chain (HC_ABD/LC_ABD) to extend their serum half-life. These modified Abs were able to bind albumin from different species in vitro. Furthermore, tumor cell lysis of IgA-Her2-LC_ABD Abs in vitro was similar to unmodified IgA-Her2 Abs. Pharmacokinetic studies in mice revealed that the serum exposure and half-life of the modified IgA-Her2 Abs was extended. In a xenograft mouse model, the modified IgA1 Abs exhibited a slightly, but significantly, improved anti-tumor response compared to the unmodified Ab. In conclusion, empowering IgA Abs with albumin-binding capacity results in in vitro and in vivo functional Abs with an enhanced exposure and prolonged half-life.     

Molecular characterization of the anti-idiotypic immune response of a relapse-free neuroblastoma patient following antibody therapy: a possible vaccine against tumors of neuroectodermal origin?

Neuroblastoma treatment with chimeric antidisialoganglioside GD2 Ab ch14.18 showed objective antitumor responses. Production of anti-idiotypic Abs (Ab2) against ch14.18 (Ab1) in some cases was positively correlated with a more favorable prognosis. According to Jerne's network theory, a subset of anti-idiotypic Abs (Ab2beta) carries an "internal image" of the Ag and induces Abs (Ab3) against the original Ag. The molecular origin of an anti-idiotypic Ab response in tumor patients was not investigated previously. To clone anti-idiotypic Abs, B cells of a ch14.18-treated neuroblastoma patient with Ab2 serum reactivity were used to construct Ab phage display libraries. After repeated biopannings on ch14.18 and its murine relative, anti-GD2 mAb 14G2a, we selected 40 highly specific clones. Sequence analysis revealed at least 10 of 40 clones with different Ig genes. Identities to putative germline genes ranged between 94.90 and 100% for V(H) and between 93.90 and 99.60% for V(L). An overall high rate of replacement mutations suggested a strong Ag-driven maturation of the anti-idiotypic Abs. Two clones that were analyzed further, GK2 and GK8, inhibited binding of ch14.18 to GD2 just as the patient's serum did. GK8 alone inhibited >80% of the patient's anti-idiotypic serum Abs in binding to ch14.18. Rabbits vaccinated with GK8 or GK2 (weaker) produced Ab3 against the original target Ag GD2. GK8 may be useful as a tumor vaccine for GD2-positive [corrected] tumors.     

Superantigen-staphylococal-enterotoxin-A-dependent and antibody-targeted lysis of GD2-positive neuroblastoma cells

 Superantigens such as the staphylococcal enterotoxin A (SEA) are among the most potent T cell activators known. They bind to major histocompatibility complex (MHC) class II molecules and interact with T cells depending on their T cell receptor (TCR) Vβ expression. Superantigens also induce a variety of cytokines and trigger a direct cytotoxic effect against MHC-class-II-positive target cells. In order to extend superantigen-dependent cell-mediated cytotoxicity (SDCC) to MHC-class-II-negative neuroblastoma cells, SEA was linked to the anti-ganglioside GD₂ human/mouse chimeric monoclonal antibody (mAb) ch14.18. Ganglioside GD₂ is expressed on most tumours of neuroectodermal origin but is expressed to a lesser extent on normal tissues. The linkage of ch14.18 to SEA was achieved either with a protein-A–SEA fusion protein or by chemical coupling. Both constructs induced T-cell-mediated cytotoxicity towards GD₂-positive neuroblastoma cells in an effector-to-target(E:T)-ratio-and dose-dependent manner in vitro. To reduce the MHC class II affinity of SEA, a point mutation was introduced in the SEA gene (SEAm9) that resulted in 1000-fold less T cell killing of MHC-class-II-expressing cells as compared to native SEA. However, a protein-A–SEAm9 fusion protein mediated cytotoxicity similar to that of protein-A–SEA on ch14.18-coated, MHC-class-II-negative neuroblastoma cells. Taken together, these findings suggest that superantigen-dependent and monoclonal-antibody-targeted lysis may be a potent novel approach for neuroblastoma therapy. Received: 15 March 1995 / Accepted: 22 May 1995     

Isotype can affect the fine specificity of an antibody for a polysaccharide antigen

Ab specificity is determined by V region sequence. The murine Mab 18B7 (IgG1) binds to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan and produces annular immunofluorescence (IF) on yeast cells. The heavy and light V regions of 18B7 were expressed with the human C regions micro, gamma 1, gamma 2, gamma 3, gamma 4, and alpha1, and the specificity and binding properties of these mouse-human chimeric (ch) Abs was determined. The chIgG1, chIgG2, chIgG4, and the chIgA produced annular IF, whereas the IgM and IgG3 produced punctate IF, despite identical V region sequences. Competition experiments with murine Abs that competed with mAb 18B7 and binding assays to peptide mimetics of glucuronoxylomannan provided additional evidence for altered specificity in some of the ch Abs. Expression of 18B7 heavy V region with murine micro C region produced IgM with a punctate IF, indicating that a change in fine specificity also accompanied the change from murine IgG1 to IgM. Our results show that Ab fine specificity can be a function of isotype. This phenomenon may be most apparent for Abs that bind to Ag with repeating epitopes, such as polysaccharides, where the quarternary structure of the Ag-Ab complex may be influenced by such constraints as Fab-Fab angles, Fc-Fc interactions, Ab size, and solvent accessibility to exposed surfaces. Alterations in Ab fine specificity following isotype change could have important implications for current concepts on the generation of secondary Ab responses to certain Ags and for the isotype preference observed in Abs to polysaccharides.     

NXS2 murine neuroblastomas express increased levels of MHC class I antigens upon recurrence following NK-dependent immunotherapy

We evaluated recurrent NXS2 neuroblastoma tumors that developed following NK- or T-cell–mediated immunotherapy in tumor-bearing mice. Recurrent tumors developed following an NK-dependent antitumor response using a suboptimal dose of hu14.18-IL2, a humanized IL-2 immunocytokine targeted to the GD₂-ganglioside. This treatment initially induced complete resolution of measurable tumor in the majority of mice, followed, however, by delayed tumor recurrence in some mice. These recurrent NXS2 tumors revealed markedly enhanced (> fivefold) MHC class I antigen expression when compared with NXS2 tumors growing in PBS-treated control mice. A similar level of enhanced MHC class I antigen-expression could be induced on NXS2 cells in vitro by culturing with interferon , and was associated with reduced susceptibility to both NK-cell–mediated tumor cell lysis and antibody-dependent cellular cytotoxicity in vitro. In contrast, Flt3-ligand treatment of NXS2-bearing mice induced a protective T-cell–dependent antitumor memory response. Recurrent NXS2 tumors that developed following Flt3-L therapy revealed a decreased expression of MHC class I antigens. While NXS2 tumors are susceptible to in vivo destruction following either hu14.18-IL2 or Flt3-ligand immunotherapies, these results suggest that some tumor cells may be selected to survive and progress by expressing either higher or lower levels of MHC class I antigen in order to resist either NK- or T-cell–mediated antitumor responses, respectively.Abbreviations ADCC antibody-dependent cellular cytotoxicity - Flt3-L Flt3-ligand - GD₂ GD₂-disialoganglioside - IC immunocytokine - mAb monoclonal antibody - NB neuroblastoma - NXS2 transplantable murine neuroblastoma - s.c. subcutaneous     

Tolerability,response and outcome of high-risk neuroblastoma patients treated with long-term infusion of anti-GD2 antibody ch14.18/CHO

Immunotherapy with short term infusion (STI) of monoclonal anti-GD₂ antibody (mAb) ch14.18 (4 × 25 mg/m²/d; 8–20 h) in combination with cytokines and 13-cis retinoic acid (RA) prolonged survival in high-risk neuroblastoma (NB) patients. Here, we investigated long-term infusion (LTI) of ch14.18 produced in Chinese hamster ovary cells (ch14.18/CHO; 10 × 10 mg/m²; 24 h) in combination with subcutaneous (s.c.) interleukin-2 (IL-2) in a single center program and report clinical response, toxicity and survival. Fifty-three high-risk NB patients received up to 6 cycles of 100 mg/m² ch14.18/CHO (d8–17) as LTI combined with 6 × 10⁶ IU/m² s.c. IL-2 (d1–5; 8–12) and 160 mg/m² oral RA (d19–32). Pain toxicity was documented with validated pain scores and intravenous (i.v.) morphine usage. Response was assessed in 37/53 evaluable patients following International Neuroblastoma Risk Group criteria. Progression-free (PFS) and overall survival (OS) was analyzed by the Kaplan-Meier method and compared to a matched historical control group from the database of AIEOP, the “Italian Pediatric Ematology and Oncology Association”. LTI of ch14.18/CHO showed acceptable toxicity profile indicated by low pain scores, reduced i.v. morphine usage and low frequency of Grade ≥3 adverse events that allowed outpatient treatment. We observed a best response rate of 40.5% (15/37; 5 CR, 10 PR), 4-year (4 y) PFS of 33.1% (observation 0.1- 4.9 y, mean: 2.2 y) and a 4 y OS of 47.7% (observation 0.27 – 5.20 y, mean: 3.6 y). Survival of the entire cohort (53/53) and the relapsed patients (29/53) was significantly improved compared to historical controls. LTI of ch14.18/CHO thus shows an acceptable toxicity profile, objective clinical responses and a strong signal of clinical efficacy in NB patients.     

Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.     

Variable-region-identical antibodies differing in isotype demonstrate differences in fine specificity and idiotype

A central tenet of the current understanding of the relationship between Ab structure and function is that the variable region domain is solely responsible for Ag specificity. However, this view was recently challenged by the observation that families of mouse-human chimeric Abs with identical V regions demonstrate differences in fine specificity and by reports of changes in Ab Id structure with isotype switching. Here we revisited this question by evaluating the reactivity of two families of murine IgG switch variants that differed in V region usage for Cryptococcus neoformans glucuronoxylomannan, glucuronoxylomannan peptide mimetics, and anti-Id mAbs. The results reveal isotype-related differences in fine specificities and Id for two mAb isotype switched families, thus establishing the validity of this observation with sets of homologous Abs. The results suggest that the C region affects V region protein conformation, leading to differences in fine specificity and Id. The finding that isotype can affect fine specificity has major implications for current concepts of the generation of secondary responses, idiotypic network regulation, and isotype function. Given that isotype class switching and Ig gene somatic hypermutation share molecular mechanisms, these observations unify these processes in the sense that both can alter specificity and affinity.     

Ch14.18 antibody produced in CHO cells in relapsed or refractory Stage 4 neuroblastoma patients

Purpose: This study aimed to assess the safety, pharmacokinetic and activity profiles of the human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 produced in Chinese hamster ovary (CHO) cells (ch14.18/CHO).

Methods: Sixteen children with recurrent/refractory neuroblastoma (median age 7.6 y) were enrolled in this Phase 1 dose-finding study. Patients received ch14.18/CHO courses of 10, 20 or 30 mg/m²/day as an eight-hour infusion over five consecutive days. Three courses at the same dose level were allowed unless disease progressed. Clearance and biodistribution of radiolabelled ch14.18/CHO in Balb/c and A/J mice were analyzed.

Results: A total of 41 ch14.18/CHO courses were given (10 × 3 courses, 5 × 2 courses, 1 × 1 course). Side effects were similar in expectedness, frequency and magnitude to those reported for ch14.18/SP2/0. The dose level of 20 mg/m²/day was confirmed. Toxicity was reversible and no treatment-related deaths occurred. In children, the peak plasma concentration was 16.51 µg/ml ± 5.9 µg/ml and the half-life was 76.91 h ± 52.5 h. A partial response following ch14.18/CHO was observed in 2/7 patients with residual disease. In mice, the half-lives were 22.7 h ± 1.9h for ch14.18/CHO and 25.0 h ± 1.9 h for ch14.18/SP2/0. The biodistribution of ¹²⁵I-ch14.18/CHO in mice with neuroblastoma was identical to ¹²⁵I-ch14.18/SP2/0, indicating GD₂ targeting activity in vivo.

Ch14.18 produced in CHO cells showed an unchanged toxicity profile and pharmacokinetics in neuroblastoma patients compared with ch14.18 produced in SP2/0 cells, and evidence of clinical activity was observed. In mice, analysis of pharmacokinetics and biodistribution showed comparable results between ch14.18/CHO and ch14.18/SP2/0. Based on these results, ch14.18/CHO was accepted for prospective clinical evaluation.     

An anti-idiotype vaccine elicits a specific response to N-glycolyl sialic acid residues of glycoconjugates in melanoma patients

We generated the 1E10 gamma-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM(3) ganglioside.     

Thymus-dependent antiidiotype and anti-antiidiotype responses to a dinitrophenyl-specific monoclonal antibody

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.     

Reduced susceptibility of recombinant polyclonal antibodies to inhibitory anti-variable domain antibody responses

The immunogenicity of therapeutic Abs is a concern as anti-drug Abs may impact negatively on the pharmacodynamics and safety profile of Ab drugs. The factors governing induction of anti-drug Abs are not fully understood. In this study, we describe a model based on mouse-human chimeric Abs for the study of Ab immunogenicity in vivo. Six chimeric Abs containing human V regions and mouse C regions were generated from six human anti-Rhesus D Abs and the Ag-binding characteristics of the parental human Abs were retained. Analysis of the immune response toward the individual chimeric Abs revealed the induction of anti-variable domain Abs including anti-idiotypic Abs against some of these, thereby demonstrating the applicability of the model for studying anti-drug Ab responses in vivo. Immunization of BALB/c, C57, and outbred NMRI mice with a polyclonal composition consisting of all six chimeric Abs demonstrated that the immunogenicity of the individual Abs was haplotype dependent. Chimeric Abs, which were nonimmunogenic when administered individually, did not become immunogenic as part of the polyclonal composition, implying the absence of epitope spreading. Ex vivo Ab-binding studies established a clear correlation between the level of immunogenicity of the Abs comprised in the composition and the impact on the pharmacology of the Abs. These analyses demonstrate that under these conditions this polyclonal Ab composition was generally less susceptible to blocking Abs than the respective mAbs.     

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.     

Antigen-specific Proteolysis by Hybrid Antibodies Containing Promiscuous Proteolytic Light Chains Paired with an Antigen-binding Heavy Chain

The antigen recognition site of antibodies consists of the heavy and light chain variable domains (V_L and V_H domains). V_L domains catalyze peptide bond hydrolysis independent of V_H domains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991) J. Biol. Chem. 266, 15571–15574). V_H domains bind antigens noncovalently independent of V_L domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544–546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the V_H domain of an anti-E2 IgG paired with promiscuously catalytic V_L domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located ∼11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit.Antibodies (Abs)² are composed of light and heavy chain subunits linked by intra- and inter-chain disulfide bonds. The noncovalent antigen binding site of Abs is formed mainly by amino acids located in the complementarity determining regions of the light and heavy chain variable domains (V_L and V_H domains). Physiological Ab-antigen binding reactions require both Ab subunits. The individual light and heavy chains can bind antigens independent of each other, but the binding affinity of the isolated subunits is often lower than the intact Abs from which they are derived (1–4). From crystallography analyses of Ab-antigen complexes, it appears that antigen contact areas with the V_H domain are somewhat greater than the V_L domain (5, 6). Recombinant IgG Abs composed of the heavy chain drawn from antigen-specific IgGs paired with irrelevant light chains retain antigen binding activity, albeit at reduced levels (1, 3).Following the initial noncovalent antigen binding step, some Abs proceed to catalyze hydrolysis of peptide bonds (7–12). The chemical catalysis step entails nucleophilic attack on the electrophilic carbonyl of peptide bonds by serine protease-like sites present in Ab V domains followed by hydrolysis of the covalent reaction intermediate if a water molecule is available (13–15). Unlike reversible binding, the catalytic function offers a means to permanently inactivate the antigen by its hydrolysis into smaller fragments. Reversibly binding Abs bind the antigen stoichiometrically (e.g. 2 antigen molecules/IgG molecule). As catalysts are reusable, a single catalytic Ab molecule can hydrolyze multiple antigen molecules. This offers the possibility of increased antigen neutralizing potency. Therefore, there is considerable interest in developing catalytic Abs directed to individual polypeptide antigens. The serine protease-like activity is a heritable trait encoded by germline Ab V genes, and Abs in the preimmune repertoire can hydrolyze peptides with diverse sequence promiscuously (13, 14, 16, 17). However, the adaptive immune system has evolved to maximize noncovalent binding affinity of Abs over the course of B cell differentiation. Physiological immune mechanisms do not favor retention and improvement of the catalytic function. B cell clonal proliferation is driven by antigen binding to B cell receptors (Abs associated with signal transducing proteins). Antigen hydrolysis by catalytic B cell receptors is followed by release of the antigen fragments, resulting in reduced B cell receptor occupancy and loss of the proliferative stimulus for the cells. Therefore, unlike the noncovalent antigen binding activity, the catalytic function is poorly selectable. Indeed, other than Abs to autoantigen and B cell superantigen substrates, there are no examples of antigen-specific catalytic Abs generated by physiological adaptive mechanisms (18).Much effort has been devoted to developing antigen-specific catalytic Abs by immune and protein engineering strategies. Based on the premise that binding to the transition state reduces the activation energy of the catalytic reaction, immunization with transition state analogs has been applied to raise Abs that catalyze ester bonds in small haptens (19). Attempts to improve the esterase activity by random mutagenesis followed by isolation of transition state analog-binding Abs have also been described (20). Developing antigen-specific proteolytic Abs, however, has been difficult because peptide bond hydrolysis is an energetically demanding reaction. Moreover, there is no viable engineering strategy available to render catalytic Abs specific for individual polypeptide antigens. We (8, 21, 22) and others (23, 24) have identified Ab light chains that hydrolyze peptide bonds promiscuously without participation from the heavy chain subunit. Disruption of the serine protease-like catalytic triad in an Ab light chain by site-directed mutagenesis was without effect on its ability to bind the polypeptide antigen by noncovalent means (13), and a discrete peptide epitope remote from the bond hydrolyzed by a proteolytic Ab preparation has been identified (25). This lead to a split-site model of proteolysis, in which distinct subsites present within the Ab combining site are responsible for initial noncovalent antigen binding and the ensuing peptide bond hydrolysis reaction (26). If this model is correct, it should be possible to develop hybrid proteolytic Abs specific for individual antigens by pairing light chains containing a promiscuous catalytic subsite with heavy chains that contribute the noncovalent subsite responsible for specific antigen binding. We describe proof-of-principle for this engineering approach using previously described catalytic light chains paired with the heavy chain of a monoclonal IgG that binds the hepatitis C virus (HCV) E2 coat protein. This protein is thought to be important in viral entry into hepatocytes and B cells by virtue of its ability to bind receptors expressed on the host cells (27, 28).     

Enhancement of antibody-dependent cytotoxicity with a chimeric anti-GD2 antibody

A mouse/human chimeric antibody (ch14.18) was developed that reacts with the disialoganglioside GD2 on the surface of tumor cells of neuroectodermal origin. ch14.18 has the constant regions of a human IgG1 antibody and was expressed in a murine hybridoma. This antibody was produced in tissue culture at concentrations up to 180 mg/liter of spent culture fluid. ch14.18 was characterized and compared to 14.G2a, a murine mAb against GD2 of IgG2a isotype derived from the same parental hybridoma as ch14.18. Scatchard plot analysis of data from saturation binding studies on M21 melanoma cells showed identical binding for ch14.18 and 14.G2a. Indirect immunofluorescence revealed the same staining pattern for ch14.18 and 14.G2a on different melanoma cell lines. Both antibodies were equally capable of targeting M21 xenografts in athymic nude mice. ch14.18- and 14.G2a-activated human C-mediated cytolysis of melanoma cell; however, ch14.18-mediated antibody-dependent cytotoxicity of human effector cells against melanoma cells 50- to 100-fold more efficiently than 14.G2a.     

Molecular and functional characterization of cynomolgus monkey IgG subclasses

Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc-FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.     

Pharmacokinetics and stability of the ch14.18–interleukin-2 fusion protein in mice

The fusion protein formed from ch14.18 and interleukin-2 (ch14.18-IL-2), shown to exhibit antitumor efficacy in mouse models, consists of IL-2 genetically linked to each heavy chain of the ch14.18 chimeric anti-GD2 monoclonal antibody. The purpose of this study was to determine the pharmacokinetics of ch14.18-IL-2 in mice and assess its stability in murine serum. Following i.v. injection, the fusion protein was found to have a terminal half-life of 4.1 h. Detection of IL-2 following injection of the ch14.18-IL-2 fusion protein showed a similar half-life, indicating that the fusion protein prolongs the circulatory half-life of IL-2. Detection of human IgG1 following injection of ch14.18-IL-2 showed a terminal half-life of 26.9 h. These data suggested that the native fusion protein is being altered in vivo, resulting in a somewhat rapid loss of detectable IL-2, despite prolonged circulation of its immunoglobulin components. In vitro incubation of the ch14.18-IL-2 fusion protein in pooled mouse serum at 37 degrees C for 48 h resulted in a loss of its IL-2 component, as detected in enzyme-linked immunosorbent assay systems and in proliferation assays. Polyacrylamide gel electrophoresis and Western blot analysis of the fusion protein incubated in mouse serum at 37 degrees C indicated that the ch14.18-IL-2 is cleaved, resulting in a loss of the 67-kDa band (representing the IL-2 linked to the IgG1 heavy chain) and the detection of a band of more than 50 kDa, slightly heavier than the IgG1 heavy chain itself. This suggests that the fusion protein is being cleaved in vitro within the IL-2 portion of the molecule. These studies show that (1) ch14.18-IL-2 prolongs the circulatory half-life of IL-2 (compared to that of soluble IL-2) and (2) the in vivo clearance of the fusion protein occurs more rapidly than the clearance of the ch14.18 antibody itself, possibly reflecting in vivo cleavage within the IL-2 portion of the molecule, resulting in loss of IL-2 activity.