Proliferating Cell Nuclear Antigen (PCNA) is a highly conserved protein essential for DNA replication, repair and scaffold functions in the cytosol. Specific inhibition of PCNA in cancer cells is an ...attractive anti-cancer strategy. ATX-101 is a first-in-class drug targeting PCNA, primarily in cellular stress regulation. Multiple in vivo and in vitro investigations demonstrated anti-cancer activity of ATX-101 in many tumor types and a potentiating effect on the activity of anti-cancer therapies. Healthy cells were less affected. Based on preclinical data, a clinical phase 1 study was initiated. Twenty-five patients with progressive, late-stage solid tumors were treated with weekly ATX-101 infusions at four dose levels (20, 30, 45, 60 mg/m
). ATX-101 showed a favorable safety profile supporting that vital cellular functions are not compromised in healthy cells. Mild and moderate infusion-related reactions were observed in 64% of patients. ATX-101 was quickly cleared from blood with elimination half-lives of less than 30 min at all dose levels, probably due to both, a quick cell penetration and peptide digestion in serum, as demonstrated in vivo. No tumor responses were observed but stable disease was seen in 70% of the efficacy population (n = 20). Further studies have been initiated to provide evidence of efficacy. Trial registration numbers: ANZCTR 375262 and ANZCTR 375319.
AFM13 is a bispecific, tetravalent chimeric antibody construct (TandAb) designed for the treatment of CD30-expressing malignancies. AFM13 recruits natural killer (NK) cells via binding to CD16A as ...immune effector cells. In this phase 1 dose-escalation study, 28 patients with heavily pretreated relapsed or refractory Hodgkin lymphoma received AFM13 at doses of 0.01 to 7 mg/kg body weight. Primary objectives were safety and tolerability. Secondary objectives included pharmacokinetics, antitumor activity, and pharmacodynamics. Adverse events were generally mild to moderate. The maximum tolerated dose was not reached. Pharmacokinetics assessment revealed a half-life of up to 19 hours. Three of 26 evaluable patients achieved partial remission (11.5%) and 13 patients achieved stable disease (50%), with an overall disease control rate of 61.5%. AFM13 was also active in brentuximab vedotin–refractory patients. In 13 patients who received doses of ≥1.5 mg/kg AFM13, the overall response rate was 23% and the disease control rate was 77%. AFM13 treatment resulted in a significant NK-cell activation and a decrease of soluble CD30 in peripheral blood. In conclusion, AFM13 represents a well-tolerated, safe, and active targeted immunotherapy of Hodgkin lymphoma. A phase 2 study is currently planned to optimize the dosing schedule in order to further improve the therapeutic efficacy. This phase 1 study was registered at www.clinicaltrials.gov as #NCT01221571.
•The bispecific, tetravalent antibody AFM13 represents a new approach engaging natural killer cells via CD16A to fight CD30+ malignancies.•AFM13 is well tolerated and active in Hodgkin lymphoma patients who received all standard therapies, including brentuximab vedotin.
Despite the several advances in the last few years into treatment of advanced lung cancer, the 5-year survival remains extremely low. New therapeutic strategies are currently under investigation, and ...immunotherapy seems to offer a promising treatment alternative. In the last decade, therapeutic cancer vaccines in lung cancer have been rather disappointing, mainly due to the lack of efficient predictive biomarkers. A better refinement of the patient population that might respond to treatment might finally lead to a success story. For the first time, the immune checkpoint inhibitors are demonstrating sustained antitumor response and improved survival and they may be the first immunotherapeutics available for patients with lung cancer.
To harness the immune system's cytotoxic capacity to fight solid tumors, we developed tetravalent, bifunctional antibodies that recognize EGFRvIII, the deletion variant III of EGFR, and either CD3 or ...CD16A on immune cells, thereby directing T cells or NK-cells to eliminate EGFRvIII+ cancer cells.Using phage display, we identified scFv antibodies that selectively bind to EGFRvIII. These highly EGFRvIII-specific scFv antibodies were substantially improved by affinity maturation achieving KDs in the 100 pM range or lower and used to construct a set of bispecific EGFRvIII-targeting TandAbs with a broad range of binding and cytotoxic properties. Mono- and bivalent binding constants, specificity for EGFRvIII and CD3 or CD16A, cytotoxic activity, and target-dependent effector cell activation were characterized in a panel of in vitro assays. TandAbs exhibited exquisite specificity towards the EGFRvIII antigen in Western Blot, SPR, ELISA, and FACS assays of EGFRvIII+ cells. No binding was observed to recombinant EGFR or to EGFR+ cells. The TandAbs apparent affinities for EGFRvIII were up to 25-fold improved relative to the monovalently binding scFvs, resulting in a KD of 11 pM for the best TandAb.EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs with high affinity for EGFRvIII were similarly potent in killing assays, displaying cytotoxicity towards EGFRvIII+ F98 glioma, transfected CHO or human DKMG cells with EC50 in the range of 1 pM – 10 pM. No cytotoxicity was observed on EGFR+ cells or EGFRvIII-negative cells demonstrating the high selectivity of EGFRvIII TandAbs for the tumor-specific EGFRvIII. Importantly, in the absence of EGFRvIII+ target cells in vitro TandAbs did not elicit T- or NK-cell activation, as demonstrated by their lack of proliferation. Binding to EGFRvIII in different solid tumor types and its absence from healthy tissues was shown by immunohistochemistry using a high affinity EGFRvIII-binding bivalent Diabody.In summary, EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs provide an opportunity to develop cytotoxic antibodies that solely target cancer, sparing normal tissues and thereby reduce the side effects associated with EGFR therapy.
Introduction: AFM13 is a CD30/CD16A bispecific tetravalent TandAb antibody that recruits and activates NK-cells by specific binding to CD16A for targeted lysis of CD30+ tumor cells. Given promising ...clinical activity and safety profile of AFM13 and proof-of-mechanism demonstrating dependence on the immune response, potential synergy of AFM13 and checkpoint modulators was evaluated.
Methods: Efficacy of AFM13 alone or in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies was assessed by in vitro cytotoxicity assays with human PBMCs or enriched NK-cells and CD30+ target cells as well as patient-derived xenograft in vivo models with autologous PBMC.
To evaluate NK-cell-mediated lysis of CD30+ lymphoma cell lines, 4 hour cytotoxicity assays were performed with PBMCs or enriched NK-cells as effector cells in the presence of suboptimal concentrations of AFM13 alone, and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies. For the in vivo model tumor fragments derived from surgical specimens of newly diagnosed patients with CD30+ Hodgkin Lymphoma were xenografted (PDX) in immuno-deficient mice. After 28 days mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes and intra-tumoral cytokines were evaluated on day 58.
Results: AFM13 as a single agent at suboptimal concentrations induced effector-to-target cell-dependent lysis of CD30+ lymphoma cells up to 40% using enriched NK-cells as effector cells in a 4 hour in vitro assay. Immune-modulating antibodies alone mediated substantially lower lysis (<25%). However, the addition of anti-PD-1 or anti-CD137 to AFM13 strongly enhanced specific lysis up to 70%, whereas the addition of anti-CTLA-4 to AFM13 showed no beneficial effect. The most impressive increase of efficacy was observed when AFM13 was applied together with a combination of anti-PD-1 and anti-CD137. In vivo, reduction of tumor growth was observed when AFM13 and anti-PD-1 were used as single agents or when AFM13 was combined with anti-CD137. Synergy was most impressive in these PDX models for the combination of AFM13 and anti-PD-1 which led to a very strong reduction of tumor size. Of note, reduction of tumor growth was strongly correlated with infiltrating NK- and T-cells and intra-tumoral cytokines.
Conclusions:
The combination trials performed with companion intra-tumoral assessment of lymphocytes and cytokines may enhance the efficacy of AFM13 in patients. This may be explained by a potential cross-talk between NK-cells and T-cell which was enhanced when AFM13 was used in combination with checkpoint modulators.
No relevant conflicts of interest to declare.
Introduction:
T-cell engaging immunotherapies such as bispecific T-cell recruiting antibodies such as the recently approved blinatumomab, or chimeric antigen receptor T-cells (CAR-T) have emerged as ...highly active therapeutics in patients with refractory or relapsed hematological malignancies such as ALL or NHL. However, a relevant number of heavily pretreated patients progress or do not respond to these novel therapies. The objective of this study was to determine whether patients´ treatment history impacts the T-cell engagement of novel immunotherapies by analyzing activity of AFM11 - a CD19-directed tandem diabody (TandAb®) construct with T-cells obtained from patients after different chemotherapeutic regimens (R-Bendamustine, R-CHOP, HD-BEAM).
Methods:
T-cells were isolated and enriched from NHL patients 4-6 weeks after different therapeutic regimens and characterized side by side with T-cells enriched from healthy volunteers by flow cytometry. The responsiveness of T-cells from NHL patients to AFM11 was compared with T-cells from healthy volunteers in proliferation and cytokine release assays. In addition, enriched T-cells were used as effector cells at limiting effector-to-target (E:T) ratios in heterologous cytotoxicity assays with NALM-6 target cells in the presence of AFM11 or an appropriate control TandAb.
Results:
We found less CD3+ cells (median 148.1 cells/µL in patient group vs. 1232.2 cells/µL in healthy donors), less NK-cells (median 35.9 in patient group vs. 217.8 cells/µL in healthy donors), and no B-cells in the selected patients. Among memory T-cells, the percentages of both central (CD45RO+/CD62L+) and effector memory (CD45RO+/CD62L-) CD4+ and CD8+ cells were significantly increased in patient PBMC. Finally, we examined the percentages of regulatory T-cells (Treg) among the CD4+ T-cells of patients and healthy donors. Overall, patients contained higher proportions of Treg. Patients who received R-Bendamustine or HD-BEAM treatment had more Treg, while the percentage of Treg in patients after R-CHOP was comparable to healthy donors.
Whereas CD8+ T-cells from patients and healthy volunteers showed similar proliferative response upon AFM11 stimulation in the presence of target cells, CD4+ T-cells from patients proliferated significantly more than CD4+ T-cells from healthy volunteers. No difference was observed among the patient groups with different prior chemotherapeutic regimens.
T-cells from patients produced significantly higher amounts of IFN-γ than T-cells of healthy donors upon stimulation with AFM11 and target cells. In contrast, the production of IL-10 was significantly lower by T-cells from patients when compared to healthy donors. Surprisingly, most of the T-cells from healthy donors and patients did not produce IL-6.
In heterologous cytotoxicity assays T-cells from healthy donors induced statistically significant higher specific lysis of NALM-6 in the presence of AFM11. There was no statistically significant difference between T-cells isolated from patients after different chemotherapies at an E:T ratio of 1:2.
However, at lower E:T ratios there was statistically significant difference among T-cells from patients after different chemotherapy regimens. T-cells isolated from patients who received R-CHOP treatment had the strongest lysis capacity compared to the two other groups. The lowest efficacy of AFM11-mediated target cell lysis was observed with T-cells from patients after treatment with HD-BEAM.
Conclusions:
In conclusion, T-cells of NHL patients after different chemotherapy regimens are reduced in number and have functional defects. However, AFM11 is able to activate patient T-cells for potent target cell lysis with similar efficacy as T-cells from healthy volunteers at higher E:T ratios. Only at limiting effector cell counts a lower efficacy was observed for T-cells from patients. T-cells from R-CHOP treated patients are the most active among the tested treatment groups and display similar responsiveness as T-cells from healthy donors. Lower activity was measured with T-cells from R-Bendamustine pretreated patients and substantial lower cytotoxic activity for T-cells from patients after HD-BEAM treatment. The correlation of these findings with in vivo response will be evaluated in an ongoing Phase I trial with AFM11.
Reusch:Affimed: Employment, Patents & Royalties: Patents. Einsele:Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau. Treder:Affimed: Employment.
Introduction: CD19 is expressed by B cells from early development through differentiation into plasma cells, and represents a validated target for the development of therapeutic antibodies to treat B ...cell malignancies such as Non Hodgkin Lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Different CD19-targeting T-cell engagers are investigated in clinical studies for the treatment of NHL or ALL, including Affimed's AFM11, a bispecific CD19/CD3 TandAb antibody, which is currently investigated in a phase 1 dose escalation study. Indeed, Affimed's bispecific tetravalent platform comprises not only T-cell engaging TandAbs with two binding sites for CD3, but also NK-cell recruiting TandAbs with two binding sites for CD16A. In the present study, Affimed's AFM11, was characterized and compared in in vitro and in vivo studies with the CD19/CD16A TandAb AFM12.
Methods: Analogous to the CD19/CD3 TandAb AFM11, a bispecific tetravalent TandAb AFM12 was constructed with two binding sites for CD19 and two sites for CD16A. Both TandAbs were characterized side by side for their biophysical properties, binding affinities to CD19+ tumor target cells and to their respective effector cells by flow cytometry. Kinetics and dose-response characteristics were evaluated in in vitro cytotoxicity assays. Potency and efficacy of both TandAbs were compared on different CD19+ tumor target cell lines using primary human effector cells. To compare the efficacy of AFM11 and AFM12 a patient-derived tumor xenograft model was developed.
Results: AFM12 mediated efficacious target cell lysis with a very fast on-set in vitro. Lysis induced by AFM11 was less efficacious (lower specific lysis than AFM12) but reproducibly more potent (lower EC50 value). In addition to the potency and efficacy of AFM11 and AFM12, different aspects of safety, such as effector cell activation in the presence and absence of target cells were investigated and will be described.
Conclusions:
Affimed's CD19/CD3 and CD19/CD16A TandAbs with identical anti-CD19 tumor-targeting domains but different effector cell-recruiting domains represent interesting molecules to study T-cell- or NK-cell-based immunotherapeutic approaches. The comparison of AFM11 and AFM12 demonstrated that AFM12-mediated lysis was fast and efficacious, whereas AFM11 showed a higher potency. In summary, the NK-cell recruiting TandAb AFM12 represents an alternative to T-cell recruiting molecules, as it may offer a different side effect profile, comparable to that of AFM13, the first NK-cell TandAb clinically investigated.
No relevant conflicts of interest to declare.
Introduction: AFM13 is a bispecific, tetravalent TandAb antibody that targets CD16A and CD30. Preclinical data demonstrated that AFM13 specifically harnessed NK cells to exert potent cytotoxic ...effects on tumor cells expressing CD30. Furthermore, AFM13 showed a favorable toxicity profile in cynomolgus monkeys. HL is characterized by the presence of CD30+ Reed-Sternberg cells and by a high medical need in the r/r setting, especially since the response to brentuximab vedotin in this patient group is mostly short. Here, we present the final results of a phase 1 clinical study in r/r HL including a focus on the pre-treatment with brentuximab vedotin.
Methods: Heavily pretreated patients (pts.) with r/r HL received stepwise escalated doses of intravenous AFM13 (0.01 to 7.0 mg/kg) weekly or 4.5 mg/kg twice weekly over 4 weeks. Primary objectives were safety and tolerability, secondary objectives included pharmacokinetics (PK), pharmacodynamics (PD), and clinical efficacy measured using Cheson criteria.
Results: 28 pts. were recruited, 24 pts. received AFM13 weekly and 4 pts. received AFM13 twice weekly. AFM13 was well tolerated with mainly mild to moderate adverse events. The maximum tolerated dose was not reached. AFM13 demonstrated activity, which was consistently more pronounced for all assessed parameters at doses ≥1.5 mg/kg (n=13). 3 of 13 pts. (23%) exhibited partial responses and disease control was reached in 10 of 13 pats. (77%). Importantly, AFM13 was also active in pts. refractory to brentuximab vedotin as most recent treatment. PK data revealed an AFM13 half-life of up to 19 hours. In peripheral blood, the portion of activated NK cells (CD69+), relative to total number of NK cells, increased up to 3-fold 24 hours after infusion compared to baseline. Kinetics of NK cell activation corresponded to kinetics of AFM13 serum levels. Soluble CD30 declined to zero at the end of treatment in almost all pts.
Conclusion: Phase 1 data indicate that AFM13 is active and well tolerated in heavily pretreated r/r HL pts. including brentuximab vedotin failures. To further enhance efficacy of AFM13 both, the dose regimen (based on PK/PD profile) and the duration of treatment, will be optimized in a phase 2 study. This study is currently in preparation by the German Hodgkin Study Group.
Topp:Affimed Therapeutics AG: Consultancy. von Tresckow:Takeda: Honoraria, Other; Novartis: Honoraria, Research Funding. Marschner:Affimed Therapeutics AG: Employment. Engert:Millenium Takeda: Consultancy; Affimed Therapeutics AG: Consultancy.
: Background/Aims: In this study the safety and efficacy of a monoclonal anti‐HBs, Tuvirumab (Mab), were investigated. Tuvirumab is a human monoclonal antibody recognizing the stable ‘a’‐determinant ...of the HBsAg. Methods: We included ten chronic hepatitis B patients: four received monotherapy, and six combination therapy with interferon alpha 2b. Results: Because the development of insoluble HBsAg–HBsAb complexes led to adverse events, the Mab dose had to be reduced in seven patients. In nine patients treatment was stopped prematurely because of lack of efficacy, i.e. neutralization of HBsAg in serum. However, temporary HBsAg levels were reduced by at least 50% in all patients; in three patients receiving combination therapy, background levels of HBsAg in serum were reached. A loss of serum HBV‐DNA was seen in three patients in the combination group, followed by HBeAg seroconversion in two patients. Conclusions: We conclude that Mab was not effective in achieving primary efficacy as assessed by neutralization of circulating HBsAg. Whether a combination of Mab with an antiviral agent that reduces the HBsAg load – and therefore minimizes the risk of adverse events – may result in clinical efficacy should be investigated.
Autoantibodies have been associated with autoimmune diseases. However, studies have identified autoantibodies in healthy donors (HD) who do not develop autoimmune disorders. Here we provide evidence ...of a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCR) in HD compared to patients with systemic sclerosis, Alzheimer's disease, and ovarian cancer. Sex, age and pathological conditions affect autoantibody correlation and hierarchical clustering signatures, yet many of the correlations are shared across all groups, indicating alterations to homeostasis. Furthermore, we identify relationships between autoantibodies targeting structurally and functionally related molecules, such as vascular, neuronal or chemokine receptors. Finally, autoantibodies targeting the endothelin receptor type A (EDNRA) exhibit chemotactic activity, as demonstrated by neutrophil migration toward HD-IgG in an EDNRA-dependent manner and in the direction of IgG from EDNRA-immunized mice. Our data characterizing the in vivo signatures of anti-GPCR autoantibodies thus suggest that they are a physiological part of the immune system.
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