Claudia De Lorenzo
Novel Human Antibodies: selection, engineering and therapeutic applications

Claudia De Lorenzo

E-mail
cladelor@unina.it
Biosketch

PhD, Associate Professor at the University of Naples "Federico II" and group Leader at CEINGE, has for a long time pursued research aims directed to the preparation, either from natural sources or through DNA engineering, and characterization, of protein drugs.
After her degree in Biological Sciences and her PhD in Biochemistry and Molecular Biology at The University of Naples “Federico II”, she has been working as a visiting fellow at the Department of Immunology, Imperial College School of Medicine, The Hammersmith Hospital, London, U.K. In June 2002 she was appointed full time researcher at the University of Naples "Federico II" and she has directed her interests towards anti-cancer immunotherapy, and her research group towards preparation and characterization of novel fully human immunoagents. In 2010 she has been appointed Associate Professor at the University of Naples "Federico II". Actually she is Full Professor in Biochemistry.
Her results have been published in 60 good peer reviewed international journals and presented at 89 national and international conferences.
She has been Principal or co-Investigator of many different Italian or European projects/grants funded by companies or by Italian Ministry of University, Italian Ministry of Health or Regione Campania. For the research activity, described above four international patents have been filed and she has been awarded a prize by Italian Foundation for cancer research "Guido Berlucchi".

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Research

Immunotherapy, based on the use of novel human mAbs endowed with antitumor or immunomodulatory activity, is an increasingly important strategy for cancer therapy. Monoclonal antibodies can be directed against Tumor Associated Antigens (TAAs), to inhibit their oncogenic function, or against Immune Checkpoints (IC), to modulate specific T cell responses against cancer.

In our laboratory novel human antitumor immunoagents have been successfully produced against the TAA ErbB2, which is a TKR, overexpressed in breast cancer and several other carcinomas. In particular, a fully human single chain antibody fragment (scFv), named Erbicin, able to bind to an epitope of ErbB2 different from those recognized by the clinically validated mAbs Trastuzumab and Pertuzumab, was isolated by phage display selection on live cells. A human anti-ErbB2 “compact antibody” (100kDa) was also generated by the fusion of Erbicin with a human IgG1 Fc, which was found to efficiently inhibit ErbB2-positive tumor growth both in vitro and in vivo with no cardiotoxic effects of Trastuzumab. As a further progress, we recently characterized Erbicin derived trispecific antibody constructs, named Tribodies, targeting three noncompeting epitopes (recognized by Erbicin, Trastuzumab and Pertuzumab) on the extracellular domain of ErbB2 or comprising a bivalent ErbB2 binding construct (derived from Erbicin) combined with an effector function in order to efficiently recruit T cells (by an anti-CD3 scFv).

These novel fusion proteins have the following advantages:

They are able to bind to a wider population of tumor cells as they recognize also epitopes present in Trastuzumab-resistant tumor cells expressing a receptor which lacks some extracellular regions, such as the oncogenic Delta 16 variant, or masked epitopes due to the interaction with other receptors such as MUC4, CD44 or other membrane surface proteins, thus providing an useful tool to overcome resistance;

They combine the different mechanisms of action of three different antibodies such as Trastuzumab, Pertuzumab and Erb-hcAb. Tri-specificity constitutes a differentiating feature providing the unique ability to combine in one single molecule three different targeting approaches.

They efficiently inhibit tumor cell growth and can provide a strong effector function by engaging T-cells (Fig.1).

Unfortunately, Triple-negative breast cancer (TNBC), accounting for aggressive breast cancers, do not express ErbB2. As an alternative, an attractive target for TNBC immunotherapy is nucleolin (NCL), a nucleocytoplasmic protein involved in the biogenesis of microRNAs strongly associated with tumor development and aggressiveness, which is selectively expressed on the surface of cancer cells, but not on their normal counterparts. We have already isolated a novel human anti-nucleolin scFv specifically binding to nucleolin-positive cells, and inhibiting their proliferation, but we are also planning to isolate other scFvs against antigens expressed on TNBC and cancer stem cells.

On the other hand, we performed a massive parallel screening of phage antibody library to obtain a large repertoire of fully human immunomodulatory antibodies against several-immune regulatory checkpoints to be used in monotherapy or in combinatorial treatments for cancer therapy. We used for the first time an innovative selection strategy on human activated lymphocytes (Fig. 2) to generate a large collection of scFvs against 10 different IC, called “Immunome Library”, from which scFvs specifically recognizing a given receptor could be pulled out by subsequent affinity selection cycles on recombinant purified proteins used as baits. By Next Generation Sequencing and bioinformatic analysis we ranked individual scFvs in each collection and identified those with the highest level of enrichment. Human IgGs from three of these collections (i.e. PD-1, PD-L1 and LAG-3) were generated and tested for their binding and biological activity. In particular, they were found to specifically bind to their targets with high affinity, to efficiently activate T cell proliferation, induce cytokine secretion and inhibit in vivo tumor growth. Interestingly, the novel isolated mAbs have comparable or even better binding affinity and biological activity than the clinically validated anti-PD-1 mAb Nivolumab. We will characterize also the mAbs specific for the other ICs in order to test them in monotherapy or in combinatorial treatments.

In order to explore the possibility to enhance the antitumor effects of the novel immunomodulatory antibodies we are currently testing these novel antibodies in appropriate combinations between them or with the anti-TAA mAbs for assessing the most potent combinatorial approach and its rapid translation into the clinic.

Despite the little information about cardiotoxicity of immunotherapies, the novel regimen of combination therapy of immunomodulatory mAbs and antibodies against tyrosine kinase receptors  might exacerbate cardiotoxic effects. Thus another aim of our research is to verify the putative cardotoxicity of combination therapies by performing preclinical studies on pro-inflammatory effects on human cardiomyocytes in vitro and on mouse models in vivo. Finally, we will use the best combination of antibodies for both their antitumor efficacy and lack of cardiotoxic effects for the construction of novel bispecific or trispecific constructs, such as the described tribodies, that could indeed include several advantages, such as the simultaneous inhibition of different targets, the increase of specificity and efficacy against tumor cells, the better pharmacokinetic and pharmacodynamic properties. Furthermore, bispecific constructs made up of immunomodulatory and anti-TAA antibodies could allow for the activation of T cells against cancer cells, as well as for the reduction of the costs of antibody production, as only one construct can provide the antitumor effects of different targeting agents.

Research lines
  1. Generation of novel human antibodies by phage display
  2. Construction of novel bispecific/trispecific constructs based on combinations of anti-TAA agents and immunomodulatory antibodies
  3. Cardiotoxicity of the anti-TAA and immunomodulatory antibodies in combinatorial treatments
Research Group
  • Margherita Passariello (PhD feb 2019), on a fellowship for one year
  • Cinzia Vetrei, PhD student
  • Stefania Ricci, undergraduated student
Most relevant publications
  1. De Lorenzo, C., Arciello, A., Cozzolino, R., Palmer, D.B., Laccetti, P., Piccoli, R., D’Alessio, G. A Fully Human Anti-tumor ImmunoRNase Selective for ErbB2-Positive Carcinomas. Cancer Res, 2004, 64, 4870-4.
  2. Gelardi T., Damiano V., Rosa R., Bianco R., Cozzolino R., Tortora G., Laccetti P., D’Alessio G., De Lorenzo C.  Two novel human anti-ErbB2 immunoagents are active on Trastuzumab-resistant tumors. Br J Cancer, 2010, 102: 513-519.
  3. Tocchetti CG, Ragone G, Coppola C, Rea D, Piscopo G, Scala S, De Lorenzo C, Iaffaioli RV, Arra C, Maurea N. Detection, monitoring, and management of trastuzumab-induced left ventricular dysfunction: an actual challenge. Eur J Heart Fail. 2012 Feb;14(2):130-7.
  4. Dario Palmieri, Timothy Richmond, Claudia Piovan, Tyler Sheetz, Nicola Zanesi, Fulvia Troise, Cindy James, Dorothee Wernicke-Jameson, Fata Nyei, Timothy J. Gordon, Francesco Salvatore, Vincenzo Coppola, Flavia Pichiorri, Claudia De Lorenzo and Carlo M. Croce. A Human anti-Nucleolin Recombinant Immunoagent for Cancer Therapy, Proc. Natl. Acad. Sci USA 2015, 112: 9418-9423.
  5. Sasso E, D'Avino C, Passariello M, D'Alise AM, Siciliano D, Esposito ML, Froechlich G, Cortese R, Scarselli E, Zambrano N, Nicosia A, De Lorenzo C. Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies. MAbs, 2018 Jul 1: 1-13.