The recombinant murine IgG2a antibody TA99, directed against a melanoma antigen, was used to study combination modalities that potentiate antibody-dependent cell cytotoxicity. As previously reported, IgG2a(TA99) was extremely efficacious in preventing the growth of B16 lung metastases. However, the same antibody mediated only minimal tumor growth retardation when used to treat established neoplastic masses. The therapeutic activity of IgG2a(TA99) could be substantially enhanced by co-administration with an antibodycytokine fusion (TA99-murine tumor necrosis factor [mTNF]), consisting of the TA99 antibody in single-chain variable fragment format fused to murine TNF. This fusion protein efficiently killed endothelial cells in vitro and displayed only minimal activity against B16 melanoma cells. In vivo, TA99-mTNF boosted the influx of natural killer cells and macrophages into B16 melanoma lesions. Therapy studies with two different administration schedules showed that the combination of TA99-mTNF and IgG2a(TA99) was superior to the individual products used as single agents. The combination treatment converted most of the tumor mass into a necrotic lesion, but a vital tumor rim eventually regrew, even when dacarbazine was included in the therapeutic regimen. The treatment modality described in this article may be applicable to the treatment of melanoma patients, given the specificity of the gp75 antigen and its conservation across species.

Colorectal cancer represents the second most common cause of cancer-related death. The human A33 transmembrane glycoprotein is a validated tumor-associated antigen, expressed in 95% of primary and metastatic colorectal cancers. Using phage display technology, we generated a human monoclonal antibody (termed A2) specific to human A33 and we compared its epitope and performance to those of previously described clinical-stage anti-human A33 antibodies. All antibodies recognized a similar immunodominant epitope, located in the V-domain of A33, as revealed by SPOT analysis. The A2 antibody homogenously stained samples of poorly, moderately, and well differentiated colon adenocarcinomas. All antibodies also exhibited an intense staining of healthy human colon sections. The A2 antibody, reformatted in murine IgG2a format, preferentially localized to A33-transfected CT26 murine colon adenocarcinomas in immunocompetent mice with a homogenous distribution within the tumor mass, while other antibodies exhibited a patchy uptake in neoplastic lesions. A2 efficiently induced killing of A33-expressing cells through antibody-dependent cellmediated cytotoxicity in vitro and was able to inhibit the growth of A33-positive murine CT26 and C51 lung metastases in vivo. Anti-A33 antibodies may thus represent useful vehicles for the selective delivery of bioactive payloads to colorectal cancer, or may be used in IgG format in a setting of minimal residual disease.

Colorectal cancer represents the second most common cause of cancer-related death. The human A33 transmembrane glycoprotein is a validated tumor-associated antigen, expressed in 95% of primary and metastatic colorectal cancers. Using phage display technology, we generated a human monoclonal antibody (termed A2) specific to A33 and we compared its epitope and performance to those of previously described clinical-stage anti-human A33 antibodies. All antibodies recognized a similar immunodominant epitope, located in the Vdomain of A33, as revealed by SPOT-analysis. The A2 antibody homogenously stained samples of poorly, moderately and well-differentiated colon adenocarcinomas. All antibodies also exhibited an intense staining of healthy human colon sections. The A2 antibody, reformatted in murine IgG2a format, preferentially localized to A33-transfected CT26 murine colon adenocarcinomas in immunocompetent mice with a homogenous distribution within the tumor mass, while other antibodies exhibited a patchy uptake in neoplastic lesions. A2 efficiently induced killing of A33-expressing cells through antibody-dependent cell-mediated cytotoxicity in vitro and was able to inhibit the growth of A33-positive murine CT26 and C51 lung metastases in vivo. Anti-A33 antibodies may thus represent useful vehicles for the selective delivery of bioactive payloads to colorectal cancer, or may be used in IgG format in settings of minimal residual disease.

Programmed cell death protein 1 (PD‐1) is an immunoregulatory target which is recognized by different monoclonal antibodies, approved for the therapy of multiple types of cancer. Different anti‐PD‐1 antibodies display different therapeutic properties and there is a pharmaceutical interest to generate and characterize novel anti‐PD‐1 antibodies. We screened multiple human antibody phage display libraries to target novel epitopes on the PD‐1 surface and we discovered a unique and previously undescribed binding specificity (termed D12) from a new antibody library (termed AMG). The library featured antibody fragments in single‐chain fragment variable (scFv) format, based on the IGHV3‐23*03 (VH) and IGKV1‐39*01 (Vκ) genes. The D12 antibody was characterized by surface plasmon resonance (SPR), cross‐reacted with the Cynomolgus monkey antigen and bound to primary human T cells, as shown by flow cytometry. The antibody blocked the PD‐1/PD‐L1 interaction in vitro with an EC50 value which was comparable to the one of nivolumab, a clinically approved antibody. The fine details of the interaction between D12 and PD‐1 were elucidated by x‐ray crystallography of the complex at a 3.5 Å resolution, revealing an unprecedented conformational change at the N‐terminus of PD‐1 following D12 binding, as well as partial overlap with the binding site for the cognate PD‐L1 and PD‐L2 ligands which prevents their binding. The results of the study suggest that the expansion of antibody library repertoires may facilitate the discovery of novel binding specificities with unique properties that hold promises for the modulation of PD‐1 activity in vitro and in vivo.

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E g l i s e p é f o n m i s t e-S u i s s e Emst Gisel et Louis Pliiss, architectes. Hans Eichenberger, ingénieur diplômé. Cette église de montagne située à Rigi-Kaltbad se compose de trois niveaux. Le niveau supérieiu* comprend l'entrée, le choeur et un espace poiu* 29 personnes assises. Le niveau intermédiaire, auquel on accède par im escalier, abrite la nef de l'église, avec 95 sièges, et un espace pour le vestiaire. Le niveau inférieur comporte un antichambre-vestibule, une salle pour la congrégation des fidèles, le bureau du pasteur et les toilettes. Ce temple est caractérisé par sa façade traitée d'une façon originale et très plastique, ainsi que par le mouvement des volumes de l'ensemble. R e f o i-m c a f-i o n cBiui>cii, Siwi-|-2zei*iancl Ernst Gisel and Louis Pliiss, architects. Hans Eichenberger, diploma engineer. This mountain church at Rigi-Kaltbad has three functional levels. The top one includes the entrance, the choir and room for 29 seats. The intermediate level, which is reached via a staircase, has the main nave of the church and provides seating facilities for 95 people, as well as a cloakroom.

Intact antibodies in the IgG format typically display only moderate in vivo activity against solid tumor masses, when their mechanism of action relies on antibody-dependent cellular cytotoxicity (ADCC). The therapeutic activity is mainly limited by the lack of immune effector cells (and most notably NK cells) in the neoplastic mass. Our group had previously reported that the therapeutic activity of Rituximab and other intact immunoglobulins could be strongly potentiated by the antibody-based targeted delivery of interleukin-2 (IL2) to the tumor environment. The combination regimen induced complete remissions of established localized lymphomas and provided long-lasting protection from disseminated lymphoma, in mouse models that featured functional NK cells. In this study, we examined the ADCC activity of TA99, a monoclonal antibody which specifically recognizes gp75, a conserved tumor-associated antigen over-expressed in both murine and human melanoma cells. In a preventive setting TA99 in murine IgG2a format completely inhibited the formation of lung metastases in mice, which had been injected intravenously with B16F10 melanoma cells. However, when the B16F10 melanoma tumor was grown subcutaneously in immunocompetent mice, the ADCC activity was not potent enough to eradicate the pre-established tumors and only a modest tumor growth inhibition was observed. A microscopic tissue distribution analysis revealed that TA99-IgG2a had selectively accumulated in the tumor mass compared to normal organs and that NK cells within the neoplastic lesions were scarce. In order to increase NK cell density at the tumor site and potentiate ADCC activity, we developed fusion proteins of the TA99 antibody with murine TNFα, IL2 and IL12. The antibody-cytokine fusion proteins were used alone and in combination with TA99-IgG2a. Combination treatment led to a marked increase of anti-cancer activity against established subcutaneous tumors. Our findings indicate that the targeted delivery of certain pro-inflammatory cytokines to the tumor site potentiates the anti-cancer properties of intact antibodies, enhancing their ability to induce ADCC. Citation Format: Patrizia Murer, Jonathan D. Kiefer, Louis Plüss, Dario Neri. Potentiating the action of antibody-dependent cell-mediated cytotoxicity with immunocytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2768.