The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit ...adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens.
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.
Abstract
Bispecific antibodies that redirect the lytic activity of cytotoxic immune effector cells, such as T- and NK cells, onto tumor cells have emerged as a highly attractive and clinically ...validated treatment modality for hematological malignancies. Advancement of this therapeutic concept into solid tumor indications, however, is hampered by the scarcity of targetable antigens that are surface-expressed on tumor cells but demonstrate only limited expression on healthy tissues. To overcome this limitation, the concept of dual-targeting, i.e. the simultaneous targeting of two tumor-expressed surface antigens with limited co-expression on non-malignant cells, with multispecific antibodies has been proposed to increase tumor selectivity of antibody-induced effector cell cytotoxicity. Here, a novel CD16A (FcγRIIIa)-directed trispecific, tetravalent antibody format, termed aTriFlex, is described, that is capable of redirecting NK cell cytotoxicity to two surface-expressed antigens. Using a BCMA/CD200-based in vitro model system, the potential use of aTriFlex antibodies for dual-targeting and selective induction of NK cell-mediated target cell lysis was investigated. Bivalent bispecific target cell binding was found to result in significant avidity gains and up to 17-fold increased in vitro potency. These data suggest trispecific aTriFlex antibodies may support dual-targeting strategies to redirect NK cell cytotoxicity with increased selectivity to enable targeting of solid tumor antigens.
Development of antibody scaffolds to directly engage cytotoxic effector cells such as T-cells for therapeutic applications is limited by the scarcity of surface antigens which are expressed ...exclusively on tumor cells and show limited or no expression on non-malignant cells. We have therefore designed a novel antibody format to selectively retarget effector cell cytotoxicity to tumor cells co-expressing two surface antigens. NK-cells play an important role in the innate immune response to multiple myeloma (MM) and are known to contribute to the efficacy of novel therapeutics. We, therefore, utilized a MM-based model system to generate proof-of concept data demonstrating antibody-mediated NK-cell retargeting to cell lines co-expressing two MM-expressed surface antigens with increased selectivity ('dual-targeting').
B-cell maturation antigen (BCMA/CD269) is widely considered to be a promising target antigen for antibody-based therapies of MM due to its almost universal expression on patient myeloma cells and its restricted surface expression on cells outside of the haematological lineage. However, low levels of expression on healthy tissue, including skin, has been reported, which could account for potential side effects associated with BCMA-targeted antibody therapies due to effector cell activation in these organs.
To increase selectivity of antibody-induced, effector cell-mediated cytotoxicity towards malignant tissue, we developed a trispecific antibody format capable of selectively engaging NK-cells through bivalent binding to CD16A (FcγRIIIa) and monovalent binding to both BCMA and CD200, a second MM-expressed surface antigen found in the majority of MM patients. Using an in vitro model system, we demonstrated that binding to BCMA+/CD200+ cell lines and the resulting increase in avidity leads to preferential lysis of antigen double-positive cells compared with antigen single-positive cells. These data suggest that dual-targeting may increase the therapeutic window compared to approaches targeting only one antigen, thereby improving safety of BCMA-directed antibody therapeutics for MM. In addition to the MM-based model system used here, the novel trispecific antibody scaffolds we have developed may be adapted to alternative target combinations within MM or in other tumor indications. Moreover, they could be used to target phenotypically distinct tumor cell clones to induce deeper and more prolonged antitumor responses. Consequently, dual-targeting of effector cells to tumors using the described antibody technology could also be applied to increase safety of T-cell engaging antibodies in the absence of exclusively tumor-expressed target antigens.
Gantke:Affimed GmbH: Employment. Weichel:Affimed GmbH: Employment. Reusch:Affimed: Employment, Patents & Royalties: Patents. Ellwanger:Affimed GmbH: Employment. Fucek:Affimed GmbH: Employment. Griep:AbCheck s.r.o.: Employment. Molkenthin:AbCheck s.r.o.: Employment. Kashala:Affimed Inc.: Employment. Treder:Affimed: Employment.
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.
Antibodies from IgM Libraries Knackmuss, Stefan; Molkenthin, Vera
Recombinant Antibodies for Immunotherapy,
07/2009
Book Chapter
IgM antibodies exist both in a pentameric soluble form and as membrane-bound monomers mainly on the surface of naïve B cells, where they are part of the antigen receptor complex. Naïve B cells, ...constituting 75% of the peripheral blood B cell repertoire in humans (Klein et al., 1997), contain the largest diversity of an individual's rearranged immunoglobulin genes. The naturally occurring antibody repertoire contains specific antibodies against various antigens. In a primary immune response, B cells expressing antigen-specific IgM molecules are activated and differentiate into antibody-producing and -secreting plasma cells. Secreted antigen-specific IgM molecules are the first immunoglobulins occurring during a primary immune response. On the other hand, so-called natural antibodies exist independently of antigenic stimulation and are thought to contribute to the first line of defense against infections (Carsetti et al., 2004; Ochsenbein & Zinkernagel, 2000) as well as malignancy (Brändlein et al., 2003).In addition to antibody-secreting plasma cells, memory B cells are generated during a primary immune response, a process that includes somatic hypermutation in the germinal centers. Most of the memory B cells have undergone a class switch and do not express IgM. In humans, however, IgM molecules with somatic mutations have been identified (Van Es et al., 1992). These somatically mutated IgM molecules contribute to an individual's immunological memory and constitute about 10% of the total peripheral blood B cell repertoire (Klein et al., 1997). IgM-expressing memory B cells protect against infections by encapsulated bacteria, and develop during the first year of life (Kruetzmann et al., 2003).
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