Generación y caracterización de fragmentos de anticuerpo recombinantes multiespecíficos para inmunoterapia del cáncer

En las últimas décadas, los anticuerpos biespecíficos (AcBs) han demostrado su potencial terapéutico en cáncer, culminando con la aprobación de blinatumomab para el tratamiento de la leucemia linfoblástica aguda (2014) y amivantamab para el del cáncer de pulmón no microcítico (2021). Sin embargo, a pesar del incremento exponencial del número de AcBs en desarrollo, su uso terapéutico ha mostrado limitaciones de eficacia y seguridad inherentes a su diseño. En este trabajo se describe y evalúa un nuevo formato de anticuerpo triespecífico (AcTs) que redirige la actividad de linfocitos T hacia células de cáncer colorrectal (CCR). El AcTs en formato TriTE (del inglés, Trispecific T-cell Engager) se encuentra formado por un scFv (del inglés, single-chain Fv) anti-CD3 flanqueado por dos dominios VHH frente a los antígenos asociados a tumor (AAT) EpCAM y EGFR. Este anticuerpo, eficientemente expresado en células de mamífero y en levaduras, es capaz de reconocer sus antígenos diana en la superficie celular, así como de desencadenar la actividad citotóxica de linfocitos T de forma específica hacia células de CCR que expresan EpCAM y/o EGFR, aunque en menor medida en las células que expresan solo uno de los AAT. In vitro, la lisis de células de CCR que expresan ambos AAT promovida por TriTE fue al menos 20 veces más potente que la de células solo EpCAM+ o EGFR+. Además, fue capaz de retrasar el crecimiento tumoral y aumentar de forma significativa la supervivencia in vivo. La ausencia de actividad citotóxica frente a células de CCR doble negativas y la actividad limitada frente a células que solo expresan uno de los AAT, apuntan hacia una mayor especificidad y un mejor perfil de seguridad de TriTE con respecto a los AcBs convencionales. Además, el reconocimiento dual de dos AAT distintos puede reducir el riesgo de escape tumoral asociado a la heterogeneidad antigénica intrínseca o a la pérdida de su expresión por la presión selectiva de las terapias basadas en AcBs..

Case Report: An EGFR-Targeted 4-1BB-agonistic Trimerbody Does Not Induce Hepatotoxicity in Transgenic Mice With Liver Expression of Human EGFR

Agonistic monoclonal antibodies (mAbs) targeting the co-stimulatory receptor 4-1BB are among the most effective immunotherapeutic agents across pre-clinical cancer models. However, clinical development of full-length 4-1BB agonistic mAbs, has been hampered by dose-limiting liver toxicity. We have previously developed an EGFR-targeted 4-1BB-agonistic trimerbody (1D8N/CEGa1) that induces potent anti-tumor immunity without systemic toxicity, in immunocompetent mice bearing murine colorectal carcinoma cells expressing human EGFR. Here, we study the impact of human EGFR expression on mouse liver in the toxicity profile of 1D8N/CEGa1. Systemic administration of IgG-based anti-4-1BB agonist resulted in nonspecific immune stimulation and hepatotoxicity in a liver-specific human EGFR-transgenic immunocompetent mouse, whereas in 1D8N/CEGa1-treated mice no such immune-related adverse effects were observed. Collectively, these data support the role of FcγR interactions in the major off-tumor toxicities associated with IgG-based 4-1BB agonists and further validate the safety profile of EGFR-targeted Fc-less 4-1BB-agonistic trimerbodies in systemic cancer immunotherapy protocols.This study was supported by grants from the European Union [IACT Project (602262)], the Spanish Ministry of Science and Innovation; the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, PID2019-110405RB-100, 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).Peer reviewe

Case Report: An EGFR-Targeted 4-1BB-agonistic Trimerbody Does Not Induce Hepatotoxicity in Transgenic Mice With Liver Expression of Human EGFR

Agonistic monoclonal antibodies (mAbs) targeting the co-stimulatory receptor 4-1BB are among the most effective immunotherapeutic agents across pre-clinical cancer models. However, clinical development of full-length 4-1BB agonistic mAbs, has been hampered by dose-limiting liver toxicity. We have previously developed an EGFR-targeted 4-1BB-agonistic trimerbody (1D8N/CEGa1) that induces potent anti-tumor immunity without systemic toxicity, in immunocompetent mice bearing murine colorectal carcinoma cells expressing human EGFR. Here, we study the impact of human EGFR expression on mouse liver in the toxicity profile of 1D8N/CEGa1. Systemic administration of IgG-based anti-4-1BB agonist resulted in nonspecific immune stimulation and hepatotoxicity in a liver-specific human EGFR-transgenic immunocompetent mouse, whereas in 1D8N/CEGa1-treated mice no such immune-related adverse effects were observed. Collectively, these data support the role of FcγR interactions in the major off-tumor toxicities associated with IgG-based 4-1BB agonists and further validate the safety profile of EGFR-targeted Fc-less 4-1BB-agonistic trimerbodies in systemic cancer immunotherapy protocols

Prospective study of diagnostic accuracy in the detection of high-grade prostate cancer in biopsy-naïve patients with clinical suspicion of prostate cancer who underwent the Select MDx test

[Objectives] This study aimed to externally validate the diagnostic accuracy of the Select MDx test for Significant prostate cancer (Sig PCa) (ISUP > 1), in a contemporaneous, prospective, multicenter cohort with a prostate-specific antigen (PSA) between 3 and 10 ng/ml and a non-suspicious digital rectal examination.[Methods and Participants] For all enrolled patients, the Select Mdx test, the risk calculator ERSPC3 + DRE, and a prostatic magnetic resonance imaging (MRI) were carried out. Subsequently, a systematic 12-core trans-rectal biopsy and a targeted biopsy, in the case of a prostate imaging–reporting and data system (PIRADS) > 2 lesion (max three lesions), were performed. To assess the accuracy of the Select MDx test in the detection of clinically Sig PCa, the test sensitivity was evaluated. Secondary objectives were specificity, negative predictive value (NPV), positive predictive value (PPV), and area under the curve (AUC). A direct comparison with the ERSPC + DRE risk calculator and MRI were also performed. We also studied the predictive ability to diagnose Sig PCa from the combination of the Select MDx test with MRI using clinical decision-curve analysis.[Results] There were 163 patients enrolled after meeting the inclusion criteria and study protocol. The Select MDx test showed a sensitivity of 76.9% (95% CI, 63.2–87.5), 49.6% specificity (95% CI, 39.9–59.2), 82.09% (95% CI, 70.8–90.4) NPV, and 41.67% (95% CI, 31.7–52.2) PPV for the diagnosis of Sig PCa. COR analysis was also performed, which showed an AUC of 0.63 (95% CI, 0.56–0.71). There were no differences in the accuracy of Select MDx, ERSPC + DRE, or MRI. The combination of Select MDX + MRI showed the highest impact in the decision-curve analysis, with an NPV of 93%.[Conclusion] Our study showed a worse performance for the SelectMdx test than previously reported, within a cohort of patients with a PSA 3–10 ng/ml and a normal DRE, with results similar to those from ERSPC + DRE RC and MRI, but with an improvement in the usual PSA pathway. A combination of the Select Mdx test and MRI could improve accuracy, but studies specifically evaluating this scenario with a cost-effective analysis are needed.This study received grant support from Ferrer to support the test performance, research meetings, and Central Registration Depository and CRO monitoring of the information platform for multicenter studies (AEU-PIEM/2018/0004).Peer reviewe

Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity

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).Peer reviewe

A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response

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)Peer reviewe

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

Bi- and trispecific immune cell engagers for immunotherapy of hematological malignancies

Abstract Immune cell engagers are engineered antibodies with at least one arm binding a tumor-associated antigen and at least another one directed against an activating receptor in immune effector cells: CD3 for recruitment of T cells and CD16a for NK cells. The first T cell engager (the anti-CD19 blinatumomab) was approved by the FDA in 2014, but no other one hit the market until 2022. Now the field is gaining momentum, with three approvals in 2022 and 2023 (as of May): the anti-CD20 × anti-CD3 mosunetuzumab and epcoritamab and the anti-B cell maturation antigen (BCMA) × anti-CD3 teclistamab, and another three molecules in regulatory review. T cell engagers will likely revolutionize the treatment of hematological malignancies in the short term, as they are considerably more potent than conventional monoclonal antibodies recognizing the same tumor antigens. The field is thriving, with a plethora of different formats and targets, and around 100 bispecific T cell engagers more are already in clinical trials. Bispecific NK cell engagers are also in early-stage clinical studies and may offer similar efficacy with milder side effects. Trispecific antibodies (engaging either T cell or NK cell receptors) raise the game even further with a third binding moiety, which allows either the targeting of an additional tumor-associated antigen to increase specificity and avoid immune escape or the targeting of additional costimulatory receptors on the immune cell to improve its effector functions. Altogether, these engineered molecules may change the paradigm of treatment for relapsed or refractory hematological malignancies

TGF-β-induced IGFBP-3 is a key paracrine factor from activated pericytes that promotes colorectal cancer cell migration and invasion.

The crosstalk between cancer cells and the tumor microenvironment has been implicated in cancer progression and metastasis. Fibroblasts and immune cells are widely known to be attracted to and modified by cancer cells. However, the role of pericytes in the tumor microenvironment beyond endothelium stabilization is poorly understood. Here, we report that pericytes promoted colorectal cancer (CRC) cell proliferation, migration, invasion, stemness, and chemoresistance in vitro, as well as tumor growth in a xenograft CRC model. We demonstrate that coculture with human CRC cells induced broad transcriptomic changes in pericytes, mostly associated with TGF-β receptor activation. The prognostic value of a TGF-β response signature in pericytes was analyzed in CRC patient data sets. This signature was found to be a good predictor of CRC relapse. Moreover, in response to stimulation by CRC cells, pericytes expressed high levels of TGF-β1, initiating an autocrine activation loop. Investigation of secreted mediators and underlying molecular mechanisms revealed that IGFBP-3 is a key paracrine factor from activated pericytes affecting CRC cell migration and invasion. In summary, we demonstrate that the interplay between pericytes and CRC cells triggers a vicious cycle that stimulates pericyte cytokine secretion, in turn increasing CRC cell tumorigenic properties. Overall, we provide another example of how cancer cells can manipulate the tumor microenvironment.This study was funded by grants from Instituto de Salud Carlos III (PI13/00090, PI16/00357), partially supported by the European Regional Development Fund (ERDF), Comunidad de Madrid (S2010‐BMD‐2312), and Ministerio de Economía y Competitividad (RTC‐2016‐5118‐1 and SAF2017‐89437‐P), cofinanced by Programa Estatal de Investigación and the European Union. AT‐G and LM‐G was supported by Comunidad Autónoma de Madrid/European Social Fund (PEJ16/MED/AI‐1961, PEJ‐2018‐PRE/BMD‐8314, and PEJ‐2018‐TL/BMD‐11483).S

Combination of T cell-redirecting strategies with a bispecific antibody blocking TGF-β and PD-L1 enhances antitumor responses

International audienceT cell-based immunotherapies for solid tumors have not achieved the clinical success observed in hematological malignancies, partially due to the immunosuppressive effect promoted by the tumor microenvironment, where PD-L1 and TGF-β play a pivotal role. However, durable responses to immune checkpoint inhibitors remain limited to a minority of patients, while TGF-β inhibitors have not reached the market yet. Here, we describe a bispecific antibody for dual blockade of PD-L1 and TFG-β, termed AxF (scFv)$_2$, under the premise that combination with T cell redirecting strategies would improve clinical benefit. The AxF (scFv)$_2$ antibody was well expressed in mammalian and yeast cells, bound both targets and inhibited dose-dependently the corresponding signaling pathways in luminescence-based cellular reporter systems. Moreover, combined treatment with trispecific T-cell engagers (TriTE) or CAR-T cells significantly boosted T cell activation status and cytotoxic response in breast, lung and colorectal (CRC) cancer models. Importantly, the combination of an EpCAMxCD3×EGFR TriTE with the AxF (scFv)$_2$ delayed CRC tumor growth in vivo and significantly enhanced survival compared to monotherapy with the trispecific antibody. In summary, we demonstrated the feasibility of concomitant blockade of PD-L1 and TGF-β by a single molecule, as well as its therapeutic potential in combination with different T cell redirecting agents to overcome tumor microenvironment-mediated immunosuppression