Cleavage of anti-PF4/heparin IgG by a bacterial protease and potential benefit in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is due to immunoglobulin G (IgG) antibodies, which bind platelet factor 4 (PF4) modified by polyanions, such as heparin (H). IgG/PF4/polyanion complexes directly activate platelets via Fc gamma type 2 receptor A (FcγRIIA) receptors. A bacterial protease, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), cleaves the hinge region of heavy-chain IgG, abolishing its ability to bind FcγR, including FcγRIIA. We evaluated whether cleavage of anti-PF4/H IgG by IdeS could suppress the pathogenicity of HIT antibodies. IdeS quickly cleaved purified 5B9, a monoclonal chimeric anti-PF4/H IgG1, which led to the formation of single cleaved 5B9 (sc5B9), without any reduction in binding ability to the PF4/H complex. However, as compared with uncleaved 5B9, the affinity of sc5B9 for platelet FcγRIIA was greatly reduced, and sc5B9 was also unable to induce heparin-dependent platelet activation. In addition, incubating IdeS in whole blood containing 5B9 or HIT plasma samples led to cleavage of anti-PF4/H antibodies, which fully abolished the ability to induce heparin-dependent platelet aggregation and tissue factor messenger RNA synthesis by monocytes. Also, when whole blood was perfused in von Willebrand factor-coated microfluidic channels, platelet aggregation and fibrin formation induced by 5B9 with heparin was strongly reduced after IdeS treatment. Finally, IdeS prevented thrombocytopenia and hypercoagulability induced by 5B9 with heparin in transgenic mice expressing human PF4 and FcγRIIA receptors. In conclusion, cleavage of anti-PF4/H IgG by IdeS abolishes heparin-dependent cellular activation induced by HIT antibodies. IdeS injection could be a potential treatment of patients with severe HIT.

Association of IgG1 Antibody Clearance with FcγRIIA Polymorphism and Platelet Count in Infliximab-Treated Patients.

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn's disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 109/L, respectively, to ≈13 days (both HR and RR) at 350 × 109/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.

The FcγRIIIA-158 VV genotype increased the risk of post-transplant lymphoproliferative disorder in T-cell-depleted kidney transplant recipients - a retrospective study.

Post-transplantation lymphoproliferative disorder (PTLD) is a severe complication in organ transplant recipients. The use of T lymphocyte-depleting antibodies (TLDAb), especially rabbit TLDAb, contributes to PTLD, and the V158F polymorphism of Fc gamma receptor IIIA (FcγRIIIA) also named CD16A could affect the concentration-effect relationship of TLDAb. We therefore investigated the association of this polymorphism with PTLD in kidney transplant recipients. We characterized the V158F polymorphism in two case-control cohorts (discovery, n = 196; validation, n = 222). Then, we evaluated the binding of rabbit IgG to human FcγRIIIA-158V and FcγRIIIA-158F. The V158F polymorphism was not linked to PTLD in the overall cohorts, but risk of PTLD was increased in VV homozygous recipients receiving TLDAb compared with F carriers in both cohorts, especially in recipients receiving TLDAb without muromonab (discovery: HR = 2.22 [1.03-4.76], P = 0.043, validation: HR = 1.75 [1.01-3.13], P = 0.049). In vitro, we found that the binding of rabbit IgG to human NK-cell FcγRIIIA was increased when cells expressed the 158-V versus the 158-F allotype. While the 158-V allotype of human FcγRIIIA binds rabbit immunoglobulin-G with higher affinity, the risk of PTLD was increased in homozygous VV kidney transplant recipients receiving polyclonal TLDAb.

CD4+ count-dependent concentration-effect relationship of rituximab in rheumatoid arthritis.

AIMS: Rituximab is approved in rheumatoid arthritis (RA). A substantial decrease in CD4+ count was observed in responders after a single cycle of treatment. This study aimed to describe and quantifying the influence of CD4+ count depletion on the concentration-response relationship of rituximab in RA patients. METHODS: In this retrospective monocentric observational study, 52 patients were assessed. Repeated measurements of rituximab concentrations (pharmacokinetics), CD4+ counts (biomarker) and disease activity score in 28 joints (DAS28, clinical response) were made. Rituximab pharmacokinetics was described using a 2-compartment model, and CD4+ cell counts and DAS28 measurements were described using indirect turnover and direct Emax pharmacokinetic-pharmacodynamic models, respectively. Delay between rituximab concentrations and responses was accounted for by including biophase compartments. RESULTS: Elimination half-life of rituximab was 18 days. The pharmacokinetic-pharmacodynamic model showed that DAS28 response to rituximab was partly associated with CD4+ cell depletion. At 6 months, a deeper DAS28 decrease was observed in patients when CD4+ cell count is decreased: median [interquartile range] of DAS28 was 3.7 [2.9-4.4] and 4.5 [3.7-5.3] in patients with and without CD4+ decrease, respectively. CONCLUSIONS: This is the first study to quantify the relationship between rituximab concentrations, CD4+ count and DAS28 in RA patients. This model showed that approximately 75% of patients had CD4+ count decrease, and that the clinical improvement is 2-fold higher in patients with CD4+ cells decrease than in others.

CMV-infected kidney grafts drive the expansion of blood-borne CMV-specific T cells restricted by shared class I HLA molecules via presentation on donor cells.

We aimed to determine the role of cytomegalovirus (CMV)-infected donor cells in the development of a CMV-specific immune response in kidney transplant recipients. We assessed the CMV pp65-specific immune response by using interferon-É£ ELISPOT and dextramers in peripheral blood mononuclear cells from 115 recipients (D+R- 31, D+R + 44, D-R + 40) late after transplantation (mean 59 ± 42 months). Receiving a kidney from a D+ donor resulted in a higher number of IFN-É£-producing anti-CMV T cells (P = .004). This effect disappeared with the absence of shared HLA class I specificities between donors and recipients (P = .430). To confirm the role of donor cells in stimulating the expansion of newly developed CMV-specific CD8+ T cells after transplantation, we compared the number of HLA-A2-restricted CMV-specific CD8+ T cells in primo-infected recipients who received an HLA-A2 or non-HLA-A2 graft. The median of anti-CMV pp65 T cells restricted by HLA-A2 was very low for patients who received a non-HLA-A2 graft vs an HLA-A2 graft (300 [0-14638] vs. 17972 [222-85594] anti-CMV pp65 CD8+ T cells/million CD8+ T cells, P = .001). This adds new evidence that CMV-infected kidney donor cells present CMV peptides and drive an inflation of memory CMV-specific CD8+ T cells, likely because of frequent CMV replications within the graft.

Increased risk of thrombosis in FcγRIIA 131RR patients with HIT due to defective control of platelet activation by plasma IgG2.

Thrombosis results in heparin-induced thrombocytopenia (HIT) from cellular activation involving Fc receptors. In this study, the FcγRIIA 131RR genotype was found to increase the risk of thrombosis in HIT patients (odds ratio: 5.9; 95% confidence interval: 1.7-20). When platelet aggregation tests (PATs) were performed with platelet-rich plasma (PRP), a shorter lag time was measured in 131RR donors compared to individuals with the HR and HH genotypes in response to HIT plasma or 5B9, a recently developed humanized monoclonal antibody to PF4/heparin. Importantly, this difference was no longer detectable when PATs were performed with washed platelets or immunoglobulin (Ig)G-depleted PRP. Moreover, polyclonal IgG or monoclonal IgG1 added to IgG-depleted PRP increased the lag time in response to 5B9. HH platelets were also sensitive to IgG2, which in contrast, failed to inhibit the response of 131RR platelets to 5B9. Finally, higher tissue factor messenger RNA levels were measured in the whole blood of 131RR donors after activation by HIT antibodies, with increased phospholipid procoagulant activity. These results demonstrate that HIT patients homozygous for the FcγRIIA 131R allele have a higher risk of thrombosis, probably due to increased cell activation by antibodies to PF4/heparin, with a lower inhibitory effect of endogenous IgG, especially from the IgG2 subclass.

Antigenic burden and serum IgG concentrations influence rituximab pharmacokinetics in rheumatoid arthritis patients.

AIMS: Rituximab is a monoclonal antibody directed against CD20, which is approved in rheumatoid arthritis (RA). This study aimed at assessing the influence of CD19+ cell counts as target-antigen amount, and of immunoglobulin G (IgG) serum concentrations on rituximab pharmacokinetics in RA patients. METHODS: In a cohort of 64 RA patients who had received repetitive courses of rituximab, the influence of CD19+ cell count, IgG serum concentration, body surface area, sex and disease activity score in 28 joints on rituximab pharmacokinetic parameters was assessed using a population pharmacokinetic analysis. RESULTS: A two-compartment model, with first-order distribution and elimination best described the data. The volume of distribution of central compartment and clearance of rituximab were estimated at 4.7 l and 0.56 l day-1 , respectively. Distribution and elimination half-lives were 0.9 days and 17.3 days, respectively. As expected, the central volume of distribution increased with body surface area (P = 0.012) and was higher in male than in female (P = 0.004). We found that the elimination rate constant (k10 ) increased with CD19+ count (P = 0.00022) and IgG concentration (P = 7.4 × 10-8 ), and that k10 decreased with time (P = 0.00015), partly explained by a change in target-antigen amount. CONCLUSIONS: The association between CD19+ count and k10 may be explained by target-mediated drug disposition, while the association between IgG serum concentration and k10 may be explained by a saturation of the neonatal Fc receptor at high IgG concentrations, resulting in decreased recycling of rituximab.

CD25 blockade in kidney transplant patients randomized to standard-dose or high-dose basiliximab with cyclosporine, or high-dose basiliximab in a calcineurin inhibitor-free regimen.

An increased basiliximab dose may saturate T-cell CD25 receptors in kidney transplant patients receiving calcineurin inhibitor (CNI)-free immunosuppression. In a 12-week study, 16 de novo kidney transplant patients were randomized to (i) 40 mg basiliximab with cyclosporine [n = 3] (controls), (ii) 80 mg basiliximab with cyclosporine [n = 6], or (iii) 80 mg basiliximab with everolimus (CNI-free) [n = 7], all with mycophenolic acid and steroids. Recruitment was stopped prematurely due to increased biopsy-proven acute rejection (BPAR) in the basiliximab 80 mg CNI-free group. BPAR occurred in 1/3, 1/6, and 4/7 patients in the three treatment groups, respectively. The primary endpoint, area under the effect curve of CD25 saturation to week 12, was 8.4(1.6) % × weeks in the control group, 11.1(1.1) % × weeks with basiliximab 80 mg + cyclosporine, and 9.7(0.7) % × weeks in the basiliximab 80 mg CNI-free group (P = 0.020 for basiliximab 80 mg + cyclosporine versus controls; P = 0.119 for basiliximab 80 mg CNI-free versus controls). Although small patient numbers prohibit robust conclusions, these results suggest that doubling the cumulative basiliximab dose to 80 mg does not provide adequate immunosuppression during the first 3 months after kidney transplantation in the absence of CNI therapy (ClinicalTrials.gov number: NCT01596062).

ADAM17-mediated shedding of FcγRIIIA on human NK cells: identification of the cleavage site and relationship with activation.

FcγRIIIA/CD16A, the low-affinity receptor for the IgG Fc portion expressed on human CD56(dim) NK cells and involved in Ab-dependent cell cytotoxicity, is shed upon NK cell activation. We found that recombinant a disintegrin and metalloprotease (ADAM) 17 cleaved the ectodomain of FcγRIIIA/CD16A and a peptide for which the sequence encompasses aa 191-201 of the FcγRIIIA/CD16A stalk region but not ADAM10. MALDI-TOF analysis revealed that the peptide was cleaved between Ala(195) and Val(196) (i.e., 1 aa upstream of the expected position). This location of the cleavage site was confirmed by the finding that ADAM17 failed to cleave a peptide in which Ala and Val were reversed. ADAM17 was found to be expressed on NK cells, and stimulation with PMA or N-ethyl-maleimide resulted in the shedding of FcγRIIIA/CD16A and CD62L, a specific substrate of ADAM17. Selective inhibition of ADAM17 prevented the shedding of both molecules. Moreover, the shedding of FcγRIIIA/CD16A was strongly correlated with degranulation when a wide range of CD56(dim) NK cell activating receptors were stimulated, whereas both ADAM17-dependent shedding and internalization were involved in FcγRIIIA/CD16A downmodulation when the latter was engaged. Finally, the shedding of FcγRIIIA/CD16A was restricted to activated cells, suggesting that ADAM17 acts mainly, if not exclusively, in cis. Taken together, our results demonstrated for the first time, to our knowledge, at the molecular level that ADAM17 cleaves the stalk region of FcγRIIIA/CD16A and identified its cleavage site. The shedding of FcγRIIIA/CD16A was at least partially ADAM17 dependent, and it may be considered as a marker of FcγRIIIA/CD16A-independent NK cell activation highly correlated with degranulation.

Use of [(18)F]-FDG PET to predict response to neoadjuvant trastuzumab and docetaxel in patients with HER2-positive breast cancer, and addition of bevacizumab to neoadjuvant trastuzumab and docetaxel in [(18)F]-FDG PET-predicted non-responders (AVATAXHER): an open-label, randomised phase 2 trial.

BACKGROUND: An effective and well tolerated treatment is needed for patients with early HER2-positive breast cancer who do not achieve a pathological complete response after neoadjuvant therapy. The AVATAXHER trial aimed to predict pathological complete response early with the use of PET and to investigate whether the addition of bevacizumab could improve the proportion of patients achieving a pathological complete response in patients unlikely to respond to treatment. METHODS: AVATAXHER was a randomised, open-label, non-comparative, multicentre phase 2 study that enrolled women (≥18 years of age) with early-stage HER2-positive breast cancer from 26 oncology centres in France. Patients initially received two cycles of neoadjuvant docetaxel (100 mg/m(2) intravenously every 3 weeks) plus trastuzumab (8 mg/kg intravenously every 3 weeks then 6 mg/kg intravenously every 3 weeks for the second course). Before the first and second cycles, [(18)F]-fluorodeoxyglucose (FDG) PET was done and the change in standardised uptake value was used to predict pathological complete response in each patient. Patients who were predicted to be responders on PET continued to receive standard therapy. Predicted non-responders were randomly assigned (2:1) to receive four cycles of docetaxel (100 mg/m(2) intravenously every 3 weeks) and trastuzumab (6 mg/kg intravenously every 3 weeks) plus bevacizumab (15 mg/kg intravenously every 3 weeks; group A) or continue on docetaxel plus trastuzumab alone (group B). Randomisation was open label and was done by an adaptive minimisation method. Although investigators and patients were aware of group assignment, the anatomo-pathologist in charge of centralised review of surgical samples and lymph nodes was masked to treatment assignment. The primary endpoint was centrally assessed pathological complete response according to the Chevallier classification. Efficacy analyses were done in the intention-to-treat population. Safety analyses in this Article were done on all patients who received at least one dose of treatment starting from cycle 3. Survival outcomes are not yet mature. This study is registered with ClinicalTrials.gov (NCT01142778) and EUDRACT (2009-013410-26). FINDINGS: Between May 19, 2010, and Oct 1, 2012, 152 patients were recruited for the study. Ten patients were subsequently excluded, leaving 142 patients in the intention-to-treat population. Of these 142 patients, 69 were predicted by [(18)F]-FDG PET to be treatment responders after two cycles of treatment. The 73 predicted non-responders were randomly assigned to group A (n=48) and group B (n=25). Pathological complete responses were noted in 37 (53·6%, 95% CI 41·2-65·7) of the PET responders, 21 (43·8%, 29·5-58·8) of those in group A, and six (24·0%, 9·4-45·1) of those in group B. Incidences of grade 3-4 adverse events were similar in all three groups. The most common grade 3-4 adverse events were neutropenia (four in PET responders, five in group A, and three in group B), febrile neutropenia (one, three, and one, respectively), and myalgia (four, none, and one, respectively). Overall, 24 serious adverse events were reported in 15 patients (PET responders: nine events in four [6%] of 67 patients; group A: 14 events in ten [21%] of 47 patients; group B: one event in one [4%] of 25 patients). No deaths occurred during the study. INTERPRETATION: In patients with HER2-positive breast cancer, early PET assessment can help to identify non-responders to neoadjuvant docetaxel plus trastuzumab therapy. In these patients, the addition of bevacizumab can increase the proportion of patients achieving a pathological complete response. This potential new role for PET and the activity of bevacizumab in this setting need to be confirmed in larger phase 3 trials. FUNDING: Roche France.

Influence of FcγRIIIA genetic polymorphism on T-lymphocyte depletion induced by rabbit antithymocyte globulins in kidney transplant patients.

INTRODUCTION: Polyclonal antithymocyte globulins (ATG) have been used in transplantation for several decades, but the sources of the interindividual variability of their effect are poorly understood. An influence of the FCGR3A-158V/F genetic polymorphism on the horse ATG concentration-effect relationship was reported in kidney transplant patients. The objective of the present study was to confirm the influence of the FCGR3A polymorphism on the extent of lymphocyte depletion in kidney transplant patients treated with rabbit antithymocyte globulin (r-ATG). MATERIALS AND METHODS: Of the 194 transplant patients treated with r-ATG between 1998 and 2002 in our institution, 69 patients were eligible and included in this retrospective study. Biomarkers of response were CD3 and CD4 counts. Dose-effect data were analyzed using a population approach, and a two-compartment turnover model with stimulation of lymphocyte 'output'. Since r-ATG concentrations were not available, a K-PD model was used. The influence of FCGR3A genotype on estimated parameters was investigated. RESULTS: The r-ATG infusion rate leading to a 50% stimulation of CD3+ output (EDK(50)), which is inversely related to patient sensitivity to r-ATG treatment, decreased with the number of V alleles (P=0.0016). CONCLUSION: The genetic polymorphism of FCGR3A influences r-ATG effect on CD3 count in kidney transplant patients, those with the V allele being more sensitive to antilymphocyte serum. These results also suggest that r-ATG act, at least in part, by antibody-dependent cellular cytotoxicity.

Risk factors for impaired CD4+ T-cell reconstitution following rabbit antithymocyte globulin treatment in kidney transplantation.

To describe long-term CD4+ T-cell reconstitution after rabbit antithymocyte globulin (rATG) treatment and identify predictive factors following kidney transplantation. A single-center retrospective study analyzed lymphocyte subsets in rATG-treated kidney transplant recipients (1986-2009). 589 patients were analyzed (maximum follow-up 21 years). A comparator group (n=298) received an anti-IL-2 receptor monoclonal antibody. CD4+ T-cell lymphopenia (<200/mm3) was present in 48.5%, 9.2%, 6.7%,2.0%, and 0% of patients at one, three, five, 10, and 20 years post-transplant, respectively. CD4+ T-cell count increased during the first 10 years but remained below the pretransplant count even after 20 years. At 1, 3, and 6 months post-transplant, mean CD4+ T-cell count was significantly lower in patients with CD4+ T-cell lymphopenia at 12 months versus patients without lymphopenia. On multivariate analyses, significant independent predictors for long-term impaired CD4 T-cell reconstitution were recipient age, pretransplant CD4+ T-cell count, 12-month CD4+ T-cell count, and tacrolimus or MMF therapy. Recipient age>40 years was identified as a cutoff point. CD4+ T-cell reconstitution following rATG treatment remains impaired even after 21 years. Most risk factors for long-term impaired CD4+ T-cell reconstitution may be evaluated pretransplant or are modifiable post-transplant.

Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.

Relationship Between Antithymocyte Globulin Concentrations and Lymphocyte Sub-Populations in Kidney Transplant Patients.

BACKGROUND: Rabbit antithymocyte globulins (rATGs) are polyclonal antibodies used to prevent acute cellular rejection in kidney transplantation. Their dosing remains largely empirical and the question of an individualized dose is still unresolved. METHODS: Data from a prospective study in 17 kidney transplant patients were used to develop a model describing the dose-concentration-response relationship of rATG with T-lymphocyte subpopulation counts over time. The model was validated using an independent cohort of kidney transplant patients treated by rATG in the same center. RESULTS: Pharmacokinetics of rATG was described using a two-compartment model integrating a third compartment and a target-mediated elimination for active rATG. The kinetics of CD3+, CD4+, CD8+, and CD3-CD56+ cell counts over time were described by a pharmacokinetic-pharmacodynamic model with transit compartments, integrating both CD3-CD56+-independent and CD3-CD56+-dependent rATG-mediated lymphocyte depletion, and a positive feedback. Elimination of rATG was influenced by age and body surface area, while its distribution was also influenced by body surface area. CD3+ proliferation rate decreased with age and CD3-CD56+-mediated elimination was influenced by the V158F-FCGR3A polymorphism. Binary efficacy and tolerance endpoints were defined as a CD3+ count < 20 mm-3 for at least 7 days and a CD4+ count > 200 mm-3 at 1 year, respectively. Simulations showed that increasing or decreasing the standard 6-mg/kg dose will impact both tolerance and efficacy, while a dose decrease may be beneficial in elderly patients. CONCLUSIONS: Our results can be used to design prospective clinical trials testing dose individualization based on patients' characteristics. CLINICAL TRIAL REGISTRATION: Eudract No. 2009-012673-35.

Anticalin N- or C-Terminal on a Monoclonal Antibody Affects Both Production and In Vitro Functionality.

Bispecific antibodies (BsAbs) represent an important advance in innovative therapeutic strategies. Among the countless formats of BsAbs, fusion with molecules such as anticalins linked to a monoclonal antibody (mAb), represents an easy and low-cost way to obtain innovative molecules. We fused an anticalin against human fibronectin to a molecule biosimilar to trastuzumab (H0) or rituximab (R0), in four different positions, two on the N terminal region of heavy or light chains and two on the C terminal region. The eight BsAbs (H family (HF) 1 to 4 and R family (RF) 1 to 4) were produced and their affinity parameters and functional properties evaluated. The presence of anticalin did not change the glycosylation of the BsAb, shape or yield. The antigenic recognition of each BsAb family, Her2 for HF1 to 4 and CD20 for RF1 to 4, was slightly decreased (HF) or absent (RF) for the anticalin N-terminal in the light chain position. The anticalin recognition of FN was slightly decreased for the HF family, but a dramatic decrease was observed for RF members with lowest affinity for RF1. Moreover, functional properties of Abs, such as CD16 activation of NK, CD32-dependent phagocytosis and FcRn transcytosis, confirmed that this anticalin position leads to less efficient BsAbs, more so for RF than HF molecules. Nevertheless, all BsAbs demonstrated affinities for CD16, CD32 and FcRn, which suggests that more than affinity for FcRs is needed for a functioning antibody. Our strategy using anticalin and Abs allows for rapid generation of BsAbs, but as suggested by our results, some positions of anticalins on Abs result in less functionality.

The Proteolytic Cleavage of Therapeutic Monoclonal Antibody Hinge Region: More Than a Matter of Subclass.

The hinge region of immunoglobulin G (IgG) is involved in C1q and FcγRIIIA-expressing natural killer (NK) cell recruitment. Both heavy chains (HCs) of the hinge region can be cleaved sequentially by several proteases of the tumor/inflammatory/infectious microenvironment, including matrix metalloproteinase 12 (MMP12), or immunoglobulin-degrading enzyme from Streptococcus pyogenes (IdeS), impairing Fc-mediated functions. The cleavage of therapeutic monoclonal antibodies (TmAbs), which are based on a human IgG1, IgG2 or IgG4 structure, has been poorly investigated, although it may represent an escape mechanism to these treatments. Therefore, we used non-reducing SDS-PAGE to compare the cleavage kinetics of five IgG1 TmAbs (trastuzumab, rituximab, cetuximab, infliximab, ipilimumab), one IgG2 TmAb (panitumumab), and two IgG4 TmAbs (nivolumab and pembrolizumab) by MMP12 and IdeS, which were found to cleave the first and second HCs with different kinetics. Panitumumab was more protease-resistant than IgG1 and IgG4 TmAbs. The latter were usually more protease-sensitive, whereas IgG1 TmAbs were usually cleaved with intermediate kinetics. However, we observed intra-subclass variability among IgG4 and IgG1 TmAbs. Nivolumab and pembrolizumab were cleaved similarly by MMP12, whereas pembrolizumab was more IdeS-resistant. Ipilimumab was more IdeS-sensitive and MMP12-resistant than the other IgG1 TmAbs, regardless of G1m allotype. In addition the Fc fragment of IgG1 TmAbs were highly resistant to cleavage by MMP12, whereas their cleavage kinetic by IdeS was very similar to that observed with the intact forms (excluding ipilimumab). Importantly, the cleavage kinetic of ipilimumab Fc fragment by IdeS was superimposable to that of trastuzumab, cetuximab and infliximab Fc fragment, showing that the variability observed for intact ipilimumab is unrelated to its Fc portion. We propose that the variability in the cleavage sensitivity/resistance balance among TmAbs of IgG1 and IgG4 subclasses results partially, from TmAb characteristics related to and/or located in the Fab region. Finally, with ELISA and flow cytometry, we observed that a single cleavage of IgG1 TmAbs greatly decreased their affinity for FcγRIIIA and C1q and their ability to induce FcγRIIIA-dependent functional responses of NK cells. Overall, our results indicate that the cleavage of the hinge region should be considered with TmAbs treatment and in the development of new molecules.

Long-term outcomes in patients with PET-predicted poor-responsive HER2-positive breast cancer treated with neoadjuvant bevacizumab added to trastuzumab and docetaxel: 5-year follow-up of the randomised Avataxher study.

BACKGROUND: The open-label, randomised Phase 2 AVATAXHER study (NCT01142778) demonstrated that early PET assessment identified HER2-positive breast cancer patients who responded poorly to neoadjuvant docetaxel plus trastuzumab. Adding neoadjuvant bevacizumab for PET-predicted poor-responders improved pathological complete response (pCR) rates (43.8% vs 24.0%). We investigated long-term study outcomes. METHODS: Patients were treated in three groups. All patients initially received two cycles of standard neoadjuvant therapy with [¹8F]-FDG PET conducted before each cycle. Those with ≥70% change in the maximum standardised uptake value (∆SUVmax) received four further cycles of standard neoadjuvant therapy (PET responders). PET-predicted poor-responders (∆SUVmax <70%) were randomised (2:1) to neoadjuvant therapy with (Group A) or without (Group B) bevacizumab for cycles 3-6. All patients received one further cycle of trastuzumab before surgery plus adjuvant trastuzumab (11 cycles). FINDINGS: 142 patients were randomized and treated (PET responders, n = 69; Group A, n = 48; Group B, n = 25). 5-year disease-free survival rates were 90.5% (95% CI: 80.0-95.6%) in PET responders, 90.2% (95% CI: 75.9-96.2%) in Group A, and 76.0% (95% CI: 54.2-88.4%) in Group B. However, no difference was observed between randomised arms in a sensitivity analysis. During adjuvant therapy, the incidence of Grade ≥3 (Group A: 25.6%; Group B 12.5%) and serious adverse events (Group A: 18.6%; Group B 12.5%) was higher in Group A vs Group B, but with no apparent effect on cardiac events. INTERPRETATION: In patients with HER2-positive breast cancer, an intervention based on early PET assessment and improvement of pCR does not modify disease-free survival. FUNDING: Roche France.

[The hinge region of therapeutic antibodies: major importance of a short sequence]. / La région charnière des anticorps thérapeutiques - L'importance capitale d'une courte séquence.

The hinge region is a short sequence of the heavy chains (H) of antibodies linking the Fab (Fragment antigen binding) region to the Fc (Fragment crystallisable) region. The functional properties of the four IgG subclasses partly result from the sequence differences of their hinge regions as some amino acids of the lower hinge region are located within or in the close vicinity of the C1q and FcγR binding sites on the IgG H chains. In addition, the hinge is susceptible to proteolytic cleavage by many proteases present in tumor and/or inflammatory microenvironment capable of affecting functional responses. Thus, an optimal format of the hinge region remains a major challenge for the development of new therapeutic antibodies.

TITLE: La région charnière des anticorps thérapeutiques - L'importance capitale d'une courte séquence. ABSTRACT: La région charnière est une courte séquence des chaînes lourdes (H) d'anticorps liant le Fab (fragment antigen binding) au Fc (fragment crystallisable). Les propriétés fonctionnelles des quatre sous-classes d'immunoglobulines d'isotype G (IgG) résultent en partie des différences de séquence de leurs régions charnières. En effet, certains acides aminés de la partie C-terminale de ces régions charnières (« partie basse ¼) sont situés au sein ou à proximité des sites de liaison de la molécule C1q de la voie classique du complément et des récepteurs pour la région Fc des IgG (RFcγ) sur les chaînes H d'IgG. Les régions charnières sont également sensibles au clivage protéolytique par de nombreuses protéases du microenvironnement tumoral et/ou inflammatoire pouvant altérer les réponses fonctionnelles. Le format optimal de la charnière reste donc un défi majeur pour le développement de nouveaux anticorps thérapeutiques.

Recombinant therapeutic monoclonal antibodies: mechanisms of action in relation to structural and functional duality.

Naked therapeutic recombinant monoclonal antibodies (mAbs) are bifunctional molecules. On the one hand, they recognize their antigen through the variable regions of the antigen binding portion (Fab). The recombinant mAb binding to a soluble or a membrane antigen may interfere with one or several functions of this antigen, leading to the therapeutic effect. On the other hand, since their crystalisable portion (Fc) is humanized (usually IgG1), they interact efficiently with human Fc-binding molecules, such as C1q and receptors for the Fc portion of IgG (FcgammaR). Thus, they initiate the classical pathway of complement and activate FcgammaR-expressing cells. The recruitment of these patient immune effector functions is essential in the therapeutic effect of several recombinant mAbs used in oncology. The aim of this review is to describe the main mechanisms of action of recombinant mAbs in relation to this structural and functional duality.

Gradual Increase of FcγRIIIa/CD16a Expression and Shift toward IFN-γ Secretion during Differentiation of CD56dim Natural Killer Cells.

Natural killer (NK) cell effector functions include cytotoxicity and secretion of cytokines such as interferon-γ (IFN-γ). The immature CD56bright subset of human NK cells lacks expression of FcγRIIIa/CD16a, one of the low-affinity immunoglobulin G receptors, or exhibits low-density expression (CD56brightCD16-/dim) and produces IFN-γ in response to cytokine stimulation, whereas the mature CD56dimCD16+ subset is the most cytotoxic one. A further differentiation/maturation of the latter subset according to the gradual loss of NKG2A and/or gain of KIR2DL (CD158a and CD158b) has been demonstrated and the ability to produce IFN-γ in response to activating receptor (AR) co-engagement is gradually acquired during terminal differentiation. In the course of flow cytometry analysis of CD56dim NK cells, we noted a substantial intraindividual heterogeneity of expression of FcγRIIIa. FcγRIIIa is unique among ARs: it does not require the co-engagement of other ARs to induce substantial cytotoxicity or cytokine synthesis in CD56dim cells. We, therefore, investigated whether individual differentiation/maturation of polyclonal CD56dim NK cells defined by expression of NKG2A/KIR2DL is related to FcγRIIIa expression and to the heterogeneity of NK cell responses upon FcγRIIIa engagement. When we analyzed unstimulated CD56dim cells by increasing level of FcγRIIIa expression, we found that the proportion of the more differentiated CD158a,h+ and/or CD158b,j+ cells and that of the less differentiated NKG2A+ cells gradually increased and decreased, respectively. FcγRIIIa engagement by using plate-bound murine anti-CD16 monoclonal antibody (mAb) or rituximab or trastuzumab (two therapeutic mAbs), resulted in donor-dependent partial segregation of IFN-γ-producing and/or degranulating CD56dim cells. Importantly, the proportion of CD158a,h/b,j+ cells and that of NKG2A+ cells was increased and decreased, respectively, IFN-γ-producing cells, whereas these proportions were poorly modified in degranulating cells. Similar results were observed after engagement of ARs by a combination of mAbs targeting NKG2D, NKp30, NKp46, and 2B4. Thus, the gradual increase of FcγRIIIa expression is an important feature of the differentiation/maturation of CD56dim cells and this differentiation/maturation is associated with a shift in functionality toward IFN-γ secretion observed upon both FcγRIIIa-dependent and FcγRIIIa-independent stimulation. The functional heterogeneity related to the differentiation/maturation of CD56dim NK cells could be involved in the variability of the clinical responses observed in patients treated with therapeutic mAbs.