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The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to −25°C. P355′s stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (kcat) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.

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).

α2-macrocroglobulin (A2M) er et protein, der findes i humant blod og besidder en unik mekanisme, hvorved det fanger og inhiberer protein-kløvende enzymer, kaldet proteaser. Mekanismen initieres ved, at en protease kløver i et bestemt område på A2M, der agerer som lokkemad. Denne kløvning får A2M til at lukke sig omkring proteasen, som derefter er indespærret og ikke længere kan kløve andre proteiner. Proteaser indvirker i kritiske biologiske processer såsom inflammation, blodkoagulation og omstrukturering af væv, og idet A2M kan inhibere de fleste humane proteaser formodes A2M også at spille en rolle i disse processer. Til trods for at der er blevet forsket i A2M siden 1950’erne, er der stadig uafklarede spørgsmål omkring dets struktur, mekanisme og biologisk funktion.Strukturen af A2M i sin native tilstand har ikke kunne løses ved traditionel røntgen krystallografi pga. dårlig spredning af røntgen stråler fra A2M krystaller. Derforhar jeg koordineret et samarbejdsstudie, hvor flere lav-opløsningsmetoder er blevetanvendt. På baggrund af studiet har vi konstrueret en model for A2M, som viser hvordan dets fire proteinkæder er sat sammen og hvordan det ser ud, når det møder proteaser, hvilket belyser den protease-hæmmende mekanisme. Modellen har en lav opløsningsgrad, men får opbakning fra flere validerende forsøg og stemmer overens med de kendte strukturer på lignende proteiner såsom komplementfaktoren C3.Jeg har igangsat produktion af rekombinant A2M og flere beslægtede proteiner i humane celler. Det har givet mulighed for at indføre ændringer i A2M for at undersøge detaljer i dets funktion. For eksempel har jeg vist, at A2Ms reaktive thiol ester gruppe kan erstattes med en disulfidbro, hvilket viser at thiol esteren ikke spiller en afgørende rolle i A2Ms mekanisme. På lignende vis kan A2Ms lokkemadsområde erstattes med enny sekvens, der ikke genkendes og kløves af humane proteaser, hvori nye kløvningssteder kan blive indsat. Denne nye sekvens blev brugt som udgangspunkt til at lave nye protease-hæmmere, der var selektive overfor bestemte proteaser.Derudover har jeg været en del af den tidlige karakterising af A2ML1, en protease-hæmmer tilhørende A2Ms proteinfamilie, som hovedsageligt er til at finde i detyderste lag af hud. Der er fundet en sammenhæng mellem genet for A2ML1 og flere sjældne sygdomme, heriblandt paraneoplastisk pemfigus, kronisk mellemørebetændelse og Noonan syndrom. Vi har opdaget at thiol esteren fra A2ML1 reagerer effektivt med hydroxyler, hvilket adskiller den fra A2M, men gør at den i i højere grad ligner komplementfaktorer som C3 og C4B. Thiol esterens hydroxyl reaktivitet viste sig at bidrage til A2ML1s hæmning af proteaser.Tilsammen har disse resultater fremmet forskningsfeltet indenfor A2M og lignende protease-hæmmere. Der er blevet grundlagt metoder og fremgange der vilgavne studier af lignende proteiner og fremtidig udvikling af A2M-baserede behandlingsformer.

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.

17 p.-4 fig.-1 tab.-1 grph. abst., Costimulation of tumor-infiltrating T lymphocytes by anti-4-1BB monoclonal antibodies (mAbs) has shown anti-tumor activity in human trials, but can be associated with significant off-tumor toxicities involving FcγR interactions. Here, we introduce albumin-fused mouse and human bispecific antibodies with clinically favorable pharmacokinetics designed to confine 4-1BB costimulation to the tumor microenvironment. These Fc-free 4-1BB agonists consist of an EGFR-specific VHH antibody, a 4-1BB-specific scFv, and a human albumin sequence engineered for high FcRn binding connected in tandem (LiTCo-Albu). We demonstrate in vitro cognate target engagement, EGFR-specific costimulatory activity, and FcRn-driven cellular recycling similar to non-fused FcRn high-binding albumin. The mouse LiTCo-Albu exhibited a prolonged circulatory half-life and in vivo tumor inhibition, with no indication of 4-1BB mAb-associated toxicity. Furthermore, we show a greater therapeutic effect when used in combination with PD-1-blocking mAbs. These findings demonstrate the feasibility of tumor-specific LiTCo-Albu antibodies for safe and effective costimulatory strategies in cancer immunotherapy., Financial support for this work was obtained from the MCIN/AEI/10.13039/501100011033 (SAF2017-89437-P and PDC2021-121711-100 to LA-V, PID2019-104544GB-I00 to CA, and PID2020-113225GB-I00 to FJB), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (PI19/00132 to LS; PI20/01030 to BB), partially supported by the ERDF; the ISCIII-RICORS within the Next Generation EU program (plan de Recuperación, Transformación y Resilencia); the Spanish Association Against Cancer (AECC 19084 to LA-V); the CRIS Cancer Foundation (FCRIS-2018-0042 and FCRIS-2021-0090 to LA-V), the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 years COVID-19 to LA-V); and the Fundació “La Caixa” (HR21-00761 project IL7R_LungCan to LA-V). AD, OAM, and KAH were funded by the Novo Nordisk Foundation, Grant; CEMBID (Center for Multifunctional Biomolecular Drug Design, Grant Number: NNF17OC0028070). OH was supported by an industrial PhD fellowship from the Comunidad de Madrid (IND2020/BMD-17668). AE-L was supported industrial PhD fellowship from the Carlos III Health Institute (IFI18/00045). CD-A was supported by a predoctoral fellowship from the Spanish Ministry of Science Innovation and Universities (PRE2018-083445). LR-P was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck. LD-A was supported by a Rio Hortega fellowship from the Carlos III Health Institute (CM20/00004).

14 p.-6 fig., Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR−EpCAM−) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity., This study was funded by grants from Instituto de Salud Carlos III PI16/00357, PI19/00132), partially supported by the European Regional Development Fund (ERDF), Comunidad Autónoma de Madrid (S2010-BMD-2312), and Ministerio de Economía y Competitividad (RTC-2016-5118-1) to L.S.; and from Ministerio de Ciencia e Innovación (SAF2017-89437-P and PID2020-117323RB-I00), partially supported by ERDF, the Spanish Association Against Cancer (AECC 19084) and the CRIS Cancer Foundation FCRIS-2018-0042, FCRIS-2021-0090 (FCRIS-2018-0042 and FCRIS-2021-0090) to L.A-V. A.T-G. was supported by a predoctoral fellowship from Comunidad Autónoma de Madrid (PEJD-2018-PRE/BMD-8314);Spanish Ministry of Science and Innovation [SAF2017-89437-P, PID2020-117323RB-I00].

Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers., L.A-V. was supported by grants from the MCIN/AEI/10.13039/ 501100011033 (PID2020-117323RB-100 and PDC2021-121711-100), the Instituto de Salud Carlos III (DTS20/00089), the CRIS Cancer Foundation (FCRIS-2021-0090), the Spanish Association Against Cancer (PROYE19084ALVA), the Fundación ‘‘La Caixa’’ (HR21-00761 project IL7R_LungCan) and the Fundación de Investigación Biomédica 12 de Octubre Programa Investiga (2022-0082). B.B and L.S. were supported by grants PI20/01030 and PI19/00132 from the Instituto de Salud Carlos III (PI20/01030). FJB and MF-G were supported by grants PID2020- 113225GB-I00 and PRE2018-085788 funded by MCIN/AEI/10.13039/ 501100011033. L.R-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck. C. D-A. was supported by a predoctoral fellowship from the MCIN/AEI/ 10.13039/501100011033 (PRE2018-083445). L.D-A. was supported by a Rio Hortega fellowship from the Instituto de Salud Carlos III (CM20/ 00004). O.H. was supported by an industrial PhD fellowship from the Comunidad de Madrid (IND2020/BMD-17668). AE-L was supported industrial PhD fellowship from the Instituto de Salud Carlos III (IFI18/ 00045)