ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy

The redirection of T cell activity using bispecific antibodies is one of the most promising cancer immunotherapy approaches currently in development, but it is limited by cytokine storm-related toxicities, as well as the pharmacokinetics and tumor-penetrating capabilities of current bispecific antibody formats. Here, we have engineered the ATTACK (Asymmetric Tandem Trimerbody for T cell Activation and Cancer Killing), a novel T cell-recruiting bispecific antibody which combines three EGFR-binding single-domain antibodies (VHH; clone EgA1) with a single CD3-binding single-chain variable fragment (scFv; clone OKT3) in an intermediate molecular weight package. The two specificities are oriented in opposite directions in order to simultaneously engage cancer cells and T cell effectors, and thereby promote immunological synapse formation. EgA1 ATTACK was expressed as a homogenous, non-aggregating, soluble protein by mammalian cells and demonstrated an enhanced binding to EGFR, but not CD3, when compared to the previously characterized tandem bispecific antibody which has one EgA1 VHH and one OKT3 scFv per molecule. EgA1 ATTACK induced synapse formation and early signaling pathways downstream of TCR engagement at lower concentrations than the tandem VHH-scFv bispecific antibody. Furthermore, it demonstrated extremely potent, dose-dependent cytotoxicity when retargeting human T cells towards EGFR-expressing cells, with an efficacy over 15-fold higher than that of the tandem VHH-scFv bispecific antibody. These results suggest that the ATTACK is an ideal format for the development of the next-generation of T cell-redirecting bispecific antibodies

Running title: Non-toxic broad anti-tumor activity of an EGFR×4-1BB bispecific trimerbod

32 p.-4 fig.Purpose: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell–mediated antitumor response. Systemic administration of anti-4-1BB–agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity.Experimental Design: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo.Results: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non–small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer.Conclusions: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.This work was supported by grants from the European Union [IACT Project (602262), H2020-iNEXT (1676)]; the Spanish Ministry of Science, Innovation and Universities and the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, CTQ2017-83810-R, RTC-2016-5118-1, RTC-2017-5944-1), partially supported by the European Regional Development Fund; the Carlos III Health Institute (PI16/00357), co-founded by the Plan Nacional de Investigación and the European Union; the CRIS Cancer Foundation (FCRIS-IFI-2018); and the Spanish Association Against Cancer (AECC, 19084). C. Domínguez-Alonso was supported by a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (PRE2018-083445). M. Zonca was supported by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness, co-founded by the European Social Fund (PTQ-16-08340).Peer reviewe

Health enhancing strength training in nonagenarians (STRONG): rationale, design and methods

<p>Abstract</p> <p>Background</p> <p>The Health Enhancing Strength Training in Nonagenarians (STRONG) is a randomised control trial to assess the effectiveness of an aerobic and strength training program for improving muscle strength, functional capacity and quality of life in nonagenarians.</p> <p>Methods</p> <p>Sixty (51 women) nonagenarians (age range: 90–102 years) who live in a geriatric nursing home will be randomly assigned to either a usual care (control) group (n = 30) or an intervention (training) group (n = 30). Participants allocated in the usual care group will receive general physical activity guidelines and participants allocated in the intervention group will also enrol in three weekly non-consecutive individualized training sessions (~45–50 min each) during 8 weeks. The exercise program will consist of muscular strength [with a special focus on leg press at 30% (start of the program) to 70% 1 repetition maximum (end)] and aerobic exercises (cycle-ergometry during 3–5 to 15 minutes at 12–14 points in the rate of perceived exertion scale).</p> <p>Results</p> <p>Results from STRONG will help to better understand the potential of regular physical activity for improving the well-being of the oldest population groups.</p> <p>Conclusion</p> <p>The increase in life expectancy together with the dramatic decrease in birth rates in industrialized countries calls the attention to health care systems and public health policymakers to focus attention on promoting healthy lifestyle in the highest sector of the population pyramid. Our study attempts to improve functional capacity and QOL of nonagenarians by implementing an individualised aerobic and strength training program in a geriatric residential care. Results from STRONG will help to better understand the potential of regular physical activity for improving the well being even in persons aged 90 years or over.</p> <p>Trail Registration</p> <p>ClinicalTrials.gov ID: NCT00848978</p

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with Fc gamma R interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8(N)/(C)EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8(N)/(C)EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8(N)/(C)EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc gamma R interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy

Generation and characterization of monospecific and bispecific hexavalent trimerbodies

Here, we describe a new class of multivalent and multispecific antibody-based reagents for therapy. The molecules, termed “trimerbodies,” use a modified version of the N-terminal trimerization region of human collagen XVIII noncollagenous 1 domain flanked by two flexible linkers as trimerizing scaffold. By fusing single-chain variable fragments (scFv) with the same or different specificity to both N- and C-terminus of the trimerizing scaffold domain, we produced monospecific or bispecific hexavalent molecules that were efficiently secreted as soluble proteins by transfected mammalian cells. A bispecific anti-laminin x anti-CD3 N-/C-trimerbody was found to be trimeric in solution, very efficient at recognizing purified plastic-immobilized laminin and CD3 expressed at the surface of T cells, and remarkably stable in human serum. The bispecificity was further demonstrated in T cell activation studies. In the presence of laminin-rich substrate, the bispecific anti-laminin x anti-CD3 N-/C-trimerbody stimulated a high percentage of human T cells to express surface activation markers. These results suggest that the trimerbody platform offers promising opportunities for the development of the next-generation therapeutic antibodies, i.e., multivalent and bispecific molecules with a format optimized for the desired pharmacokinetics and adapted to the pathological context.Ministerio de Ciencia e Innovación e InnovaciónMinisterio de Economía y CompetitividadComunidad de MadridFondo de Investigación Sanitaria/Instituto de Salud Carlos IIIEuropean Social FundFundación de Investigación Biomédica-Hospital Universitario Puerta de HierroDepto. de Bioquímica y Biología MolecularFac. de FarmaciaTRUEpu

Short-term, light- to moderate-intensity exercise training improves leg muscle strength in the oldest old: a randomized controlled trial

Our objective was to assess the effects of an 8-week exercise training program with a special focus on light- to moderate-intensity resistance exercises (30-70% of one repetition maximum, 1RM) and a subsequent 4-week training cessation period (detraining) on muscle strength and functional capacity in participants aged 90 and older. We studied a randomized controlled trial performed during March to September 2009. Forty nonagenarians (90-97) were randomly assigned to an intervention or control group (16 women and 4 men per group). We analyzed eight-week muscle strength exercise intervention focused on lower limb strength exercises of light to moderate intensity. we studied handgrip strength, 8-m walk test, 4-step stairs test, Timed Up and Go test, and number of falls. A significant group by time interaction effect (P=.02) was observed only for the 1RM leg press. In the intervention group, 1RM leg press increased significantly with training by 10.6 kg [95% confidence interval (CI)=4.1-17.1 kg; P=.01]. Except for the mean group number of falls, which were 1.2 falls fewer per participant in the intervention group (95% CI=0.0-3.0; P=.03), no significant training effect on the secondary outcome measures was found. In conclusion, exercise training, even of short duration and light to moderate intensity, can increase muscle strength while decreasing fall risk in nonagenarians.3.737 JCR (2011) Q1, 9745 Geriatrics & gerontologyUE

Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains

Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIEXVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIEXVIII modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas.FJB was supported by a grant from the Ministerio de Economía y Competitividad (CTQ2014-56966-R). JB and BO were supported by a grant from the Ministerio de Economía y Competitividad (BIO2011-22568). AA-C and AB-T were supported by Programa Torres Quevedo from Ministerio de Economía y Competitividad, cofounded by the European Social Fund (PTQ09-01-01089 and PTQ11–04604, respectively). SLH is a recipient of the 2016 Novo Scholarship