A challenge when developing therapeutic antibodies is the identification of candidates with favorable pharmacokinetics (PK) early in development. A key determinant of immunoglobulin (IgG) serum ...half‑life in vivo is the efficiency of pH-dependent binding to the neonatal Fc receptor (FcRn). Numerous studies have proposed techniques to assess FcRn binding of IgG-based therapeutics in vitro, enabling prediction of serum half-life prior to clinical assessment. FcRn high-performance liquid chromatography (HPLC) assays FcRn binding of therapeutic IgGs across a pH gradient, allowing the correlation of IgG column retention time to the half‑life of a therapeutic IgG in vivo. However, as FcRn retention time cannot be directly compared to an in vivo parameter, modifications to FcRn-HPLC are required to enable interpretation of the data within a physiological context, to provide more accurate estimations of serum half-life. This study presents an important modification to this method, FcRn-pH-HPLC, which reproducibly measures FcRn dissociation pH, allowing correlation with previously established half-lives of therapeutic antibodies. Furthermore, the influence of incorporating various antibody modifications, binding modules, and their orientations within IgGs and bispecifics on FcRn dissociation pH was evaluated using antibodies from the redirected optimized cell killing (ROCK®) platform. Target and effector antigen-binding domain sequences, their presentation format and orientation within a bispecific antibody alter FcRn retention; tested Fc domain modifications and incorporating stabilizing disulfide bonds had minimal effect. This study may inform the generation of mono-, bi- and multi-specific antibodies with tailored half-lives based on FcRn binding properties in vitro, to differentiate antibody-based therapeutic candidates with optimal developability.
Epidermal growth factor receptor (EGFR)-targeted cancer therapy such as anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors have demonstrated clinical efficacy. However, there remains a ...medical need addressing limitations of these therapies, which include a narrow therapeutic window mainly due to skin and organ toxicity, and primary and secondary resistance mechanisms of the EGFR-signaling cascade (e.g., RAS-mutated colorectal cancer). Using the redirected optimized cell killing (ROCK®) antibody platform, we have developed AFM24, a novel bispecific, IgG
-scFv fusion antibody targeting CD16A on innate immune cells, and EGFR on tumor cells. We herein demonstrate binding of AFM24 to CD16A on natural killer (NK) cells and macrophages with
values in the low nanomolar range and to various EGFR-expressing tumor cells. AFM24 was highly potent and effective for antibody-dependent cell-mediated cytotoxicity via NK cells, and also mediated antibody-dependent cellular phagocytosis via macrophages
. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status.
, AFM24 was well tolerated up to the highest dose (75 mg/kg) when administered to cynomolgus monkeys once weekly for 28 days. Notably, skin and other toxicities were not observed. A transient elevation of interleukin-6 levels was detected at all dose levels, 2-4 hours post-dose, which returned to baseline levels after 24 hours. These results emphasize the promise of bispecific innate cell engagers as an alternative cancer therapy and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status.
ADA: antidrug antibody; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; AUC: area under the curve; CAR: chimeric-antigen receptor; CD: Cluster of differentiation; CRC :colorectal cancer; ECD: extracellular domain; EGF: epidermal growth factorEGFR epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment, crystallizableFv variable fragment; HNSCC: head and neck squamous carcinomaIL interleukinm; Ab monoclonal antibody; MOA: mechanism of action; NK :natural killer; NSCLC: non-small cell lung cancer; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PD: pharmacodynamic; ROCK: redirected optimized cell killing; RSV: respiratory syncytial virus; SABC: specific antibody binding capacity; SD: standard deviation; TAM: tumor-associated macrophage; TKI: tyrosine kinase inhibitor; WT: wildtype.
Redirection of immune cells to efficiently eliminate tumor cells holds great promise. Natural killer cells (NK), macrophages, or T cells are specifically engaged with target cells expressing markers ...after infection or neoplastic transformation, resulting in their activation and subsequent killing of those targets. Multiple strategies to redirect immunity have been developed in the past two decades, but they have technical hurdles or cause undesirable side-effects, as exemplified by the T cell-based chimeric antigen receptor approaches (CAR-T therapies) or bispecific T cell engager platforms. Our first-in-class bispecific antibody redirecting innate immune cells to tumors (AFM13, a CD30/CD16A-specific innate immune cell engager) has shown signs of clinical efficacy in CD30-positive lymphomas and the potential to be safely administered, indicating a wider therapeutic window compared to T cell engaging therapies. AFM13 is the most advanced candidate from our fit-for-purpose redirected optimized cell killing (ROCK®) antibody platform, which comprises a plethora of CD16A-binding innate immune cell engagers with unique properties. Here, we discuss aspects of this modular platform, including the advantages of innate immune cell engagement over classical monoclonal antibodies and other engager concepts. We also present details on its potential to engineer a fit-for-purpose innate immune cell engager format that can be equipped with unique CD16A domains, modules that influence pharmacokinetic properties and molecular architectures that influence the activation of immune effectors, as well as tumor targeting. The ROCK® platform is aimed at the activation of innate immunity for the effective lysis of tumor cells and holds the promise of overcoming limitations of other approaches that redirect immune cells by widening the therapeutic window.
To improve recruitment and activation of natural killer (NK) cells to lyse tumor cells, we isolated a human anti-CD16A antibody with similar affinity for the CD16A 158F/V allotypes, but no binding to ...the CD16B isoform. Using CD16A-targeting Fv domains, we constructed a tetravalent bispecific CD30/CD16A tandem diabody (TandAb®) consisting solely of Fv domains. This TandAb has two binding sites for CD16A and two for CD30, the antigen identifying Hodgkin lymphoma cells. The binding and cytotoxicity of the TandAb were compared with antibodies with identical anti-CD30 domains: (1) a native IgG, (2) an IgG optimized for binding to Fc receptors, and (3) a bivalent bispecific CD30/CD16A diabody. Due to its CD16A-bivalency and reduced k
off
, the TandAb was retained longer on the surface of NK cells than the IgGs or the diabody. This contributed to the higher potency and efficacy of the TandAb relative to those of the other anti-CD30 antibodies. TandAb cytotoxicity was independent of the CD16A allotype, whereas the anti-CD30 IgGs were substantially less cytotoxic when NK cells with low affinity CD16A allotype were employed. TandAb activation of NK cells was strictly dependent on the presence of CD30
+
target cells. Therefore, the CD30/CD16A TandAb may represent a promising therapeutic for the treatment of Hodgkin's lymphoma; further, anti-CD16A TandAbs may function as potent immunotherapeutics that specifically recruit NK cells to destroy cancer cells.
Innate cell engager (ICE
) constructs are bispecific tetravalent antibodies targeting specific tumor antigens and simultaneously engaging natural killer (NK) cell and macrophage receptors for the ...destruction of tumor cells. Pre-complexing of ICE
constructs with adoptive NK cells is a novel approach to enhance NK cell activity. The suitability of such complexes for cryopreservation, whilst retaining the biological activity and specificity, may enable the development of off-the-shelf NK cell products. This study investigates the binding affinity of ICE
constructs targeting EpCAM and NK cell receptors CD16A, NKG2D, or NKp46 to the corresponding antigens, the ICE
antitumor activity, and feasibility of cryopreservation. Cell surface retention assays using primary NK cells confirmed a substantially slower ICE
construct dissociation kinetics compared with control molecules, suggesting the formation of durable complexes independently of the CD16A polymorphism. The high-affinity NK cell and EpCAM/CD16A ICE
complexes were superior to those engaging NKG2D or NKp46 receptors when tested for the NK-cell-mediated elimination of EpCAM-expressing tumor cells. Moreover, the potency and efficacy of these complexes were unaffected after a single freeze-thaw cycle. CD16A-selective ICE
drug candidates complexed with NK cells hold promise as novel cryopreserved off-the-shelf NK cell products with chimeric antigen receptor-like NK cell properties, capable of effective depletion of tumor cells.
BackgroundThe use of bispecific Innate Cell Engagers (ICE®) has become a successful strategy for immune cell activation and killing of tumor cells through antibody-dependent cellular cytotoxicity ...(ADCC).1 Combination of adoptive NK cell therapy with ICE® molecules significantly improved tumor-targeting and has shown unprecedented clinical response rates in heavily pretreated cancer patients.2 Alternatively, as NK cells alone show short time clinical activity only, genetic modification with chimeric antigen receptors (CAR) has demonstrated improved clinical success in patients with CD19+ haematological disorders.3 4 To compare both approaches, we have evaluated the efficacy of NK cells combined with tetravalent bispecific CD16A/CD19-targeting ICE® versus anti-CD19 CAR-NK cells in a preclinical proof-of-concept study using CD19-positive target cells.MethodsNK cells from PBMCs of healthy donors were expanded for three weeks in the presence of IL-2 and IL-15. Anti-CD19 CAR-NK cells were generated by transduction with baboon envelope pseudo-typed gamma-retroviral vectors. NK cell cytotoxic activation was assessed by calcein-release assays, degranulation, cytokine production, and specific CD19 positive target cell killing resolved over time using IncuCyte imaging system after co-culture of non-transduced NK cells plus CD16A/CD19-targeting ICE or anti-CD19 CAR-NK cells with CD19-positive or negative tumor target cells.ResultsCombination of non-transduced NK cells with the CD16A/CD19 ICE® enhanced ADCC activity towards CD19-positive target cells and mediated elevated levels of degranulation when compared to NK cells in the absence of ICE®. In addition, this combination induced target cell dependent TNF-alpha and IFN-gamma secretion by NK cells. CD16A/CD19 ICE-induced NK cell maximal cytotoxicity as well as TNF-alpha and IFN-gamma secretion were comparable to that of a corresponding CD19 CAR NK cells at different effector to target ratios.ConclusionsNo major difference in cytotoxicity and TNF-alpha and IFN-gamma release between the two approaches, NK cells in combination with CD16A/CD19 ICE vs. CD19 CAR NK cells, has been observed. Therefore, combination of allogeneic NK cells with bispecific ICE® represent a versatile innovation providing advantages over engineered CAR-NK cells such as more cost-effective manufacturing and potentially reduced safety concerns. Treatment options for ICE® molecules are more flexible since no genetic engineering of NK cells is needed and can be built basically for any target. Moreover, this approach is applicable to a variety of NK cell products of different origins.ReferencesPinto, et al. Reimagining antibody-dependent cellular cytotoxicity in cancer: the potential of natural killer cell engagers. Trends in Immunology 2022;43(11):932–46.Kerbauy, et al. Combining AFM13, a Bispecific CD30/CD16 Antibody, with Cytokine-Activated Blood and Cord Blood-Derived NK Cells Facilitates CAR-like Responses Against CD30+ Malignancies. Clin Cancer Res 2021;27(13):3744–3756.Gong, et al. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. Hematol Oncol 2021;14:73.Liu, et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl J Med 2020;382:545–553.Ethics ApprovalThis work was performed with NK cells from healthy anonymous blood donors approved by the State Chamber of Physicians of Saxony, Germany, under ethical vote number EK-BR-79/21–1.
To harness the cytotoxic capacity of immune cells for the treatment of solid tumors, we developed tetravalent, bispecific tandem diabody (TandAb) antibodies that recognize EGFRvIII, the deletion ...variant III of the epidermal growth factor receptor (EGFR), and CD3 on T-cells, thereby directing immune cells to eliminate EGFRvIII-positive tumor cells. Using phage display, we identified scFv antibodies selectively binding to EGFRvIII. These highly EGFRvIII-specific, fully human scFv were substantially improved by affinity maturation, achieving
s in the picomolar range, and were used to construct a set of bispecific EGFRvIII-targeting TandAbs with a broad range of binding and cytotoxic properties. These antibodies exhibited an exquisite specificity for a distinguished epitope in the N-terminal portion of EGFRvIII, as shown on recombinant antigen in Western Blot, SPR, and ELISA, as well as on antigen-expressing cells in FACS assays, and did not bind to the wild-type EGFR. High-affinity EGFRvIII/CD3 TandAbs were most potent in killing assays, displaying cytotoxicity toward EGFRvIII-expressing CHO, F98 glioma, or human DK-MG cells with EC
values in the range of 1-10 pM
. They also demonstrated dose-dependent growth control
in an EGFRvIII-positive subcutaneous xenograft tumor model. Together with the tumor-exclusive expression of EGFRvIII, the EGFRvIII/CD3 TandAbs' high specificity and strictly target-dependent activation with no off-target activity provide an opportunity to target tumor cells and spare normal tissues, thereby reducing the side effects associated with other anti-EGFR therapies. In summary, EGFRvIII/CD3 TandAbs are highly attractive therapeutic antibody candidates for selective immunotherapy of EGFRvIII-positive tumors.
To harness the potent tumor-killing capacity of T cells for the treatment of CD19
+
malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting ...solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19
+
cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19
+
malignancies with an advantageous safety risk profile and anticipated dosing regimen.
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