Search

Figure S1: Characterization of binding properties and thermal stability of Fynomer C12; Figure S2: Results of binding studies with C12-pertuzumab bispecific FynomAbs; Figure S3: Additional in vitro tumour growth inhibition studies with COVA208 on more HER2 positive cancer cell lines; Figure S4: Additional data on the induction of apoptosis in NCI-N87 cells by COVA208: dose-response curve of Caspase 3/7 activity, and Tunel assay; Figure S5: Expression data (heat map) of signalling mediators in response to treatment with COVA208, pertuzumab, or trastuzumab (RPPA analysis); Supplementary Table 1 listing expression data of 167 signaling proteins (phospho- and total proteins) in response to COVA208, pertuzumab or trastuzumab treatment of NCI-N87 cells in vitro.

Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2-targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. On the basis of the approved antibody pertuzumab, we have created a panel of bispecific FynomAbs, which target two epitopes on HER2. FynomAbs are fusion proteins of an antibody and a Fyn SH3–derived binding protein. One bispecific FynomAb, COVA208, was characterized in detail and showed a remarkable ability to induce rapid HER2 internalization and apoptosis in vitro. Moreover, it elicited a strong inhibition of downstream HER2 signaling by reducing HER2, HER3, and EGFR levels in vitro and in vivo. Importantly, COVA208 demonstrated superior activity in four different xenograft models as compared with the approved antibodies trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets. Mol Cancer Ther; 13(8); 2030–9. ©2014 AACR.

The Araris site-specific and one-step peptide linker conjugation technology generates stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. We generated an anti-Nectin-4 ADC that shows superior anti-tumor activity and tolerability compared to enfortumab-vedotin (EV) in head-to-head in vitro and in vivo studies. The Araris ADC is based on enfortumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload. Using a peptide linker and site-specific enzymatic conjugation approach, we generated a pure ADC with a drug-to-antibody-ratio (DAR) of approximately 2 and above 98 percent monomeric content. The Araris ADC demonstrated potent cell cytotoxicity similar to the approved enfortumab-vedotin which has a DAR of 4, excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while EV showed significant payload deconjugation. Despite high stability, the Araris ADC releases the free active MMAE metabolite at comparable rate to EV in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. The ADC was also shown to be extremely stable in circulation in pharmacokinetic studies in rodents, leading to an intact ADC exposure profile comparable to the unmodified enfortumab parent antibody. No free payload was detectable in circulation during the 3 week study by LCMS-MRM. In efficacy studies using a SUM-190PT established breast cancer model, a single injection at a dose of 10 ug/kg normalized by payload induced a complete tumor regression lasting for more than 100 days (i.e. a very durable response or tumor eradication). EV administered at the same payload dose showed only a short and transient (until day 20 only) tumor regression with no animal (0/6) reaching a complete response. Despite the higher in vivo exposure and extremely efficient anti-tumor response at low payload doses, there was no increased toxicity but in contrast, overall tolerability was improved, i.e., less neutropenia, skin involvement and signs of toxicity - the skin toxicity being the dose-limiting toxicity of Enfortumab vedotin in humans and rats. Overall, the highest non-severely toxic dose (HNSTD) in 4-week repeat dose rat toxicity studies for the Araris ADC (25 mg/kg) was 5-fold higher compared to the HNSTD (5mg/kg) reported for Enfortumab vedotin. Our data impressively show that the Araris ADC has superior efficacy and durable anti-tumor response even at 3-fold lower payload dose compared to EV. The improved efficacy in mice and tolerability in rates resulted in a 8-fold better TI for the Araris ADC and offers the opportunity to develop a highly efficacious ADC having potentially lower dose-limiting toxicities such as peripheral neuropathy, rashes or neutropenia. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Emma Renard, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB221.

The Araris’ site-specific and one-step linker conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-HER2 ADC using a Topoisomerase 1 (Topo1) inhibitor as payload with highly favorable biophysical properties and superior anti-tumor efficacy compared to Trastuzumab deruxtecan in head-to-head in vitro and in vivo studies. Based on trastuzumab as the targeting antibody and a Topoisimerase 1 inhibitor as payload, we generated highly homogeneous and pure ADCs with a drug-to antibody-ratio (DAR) of 2. In in-vitro assays on target positive cell-lines, the Araris Topo 1 ADC demonstrated potent cell-cytotoxicity in the low nM-range similar to the approved Trastuzumab deruxtecan which has a DAR of 8. Moreover, the ADC showed excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while Trastuzumab deruxtecan showed significant payload loss during the 14d incubation period. Interestingly, despite the improved stability, the kinetics for payload release was highly efficient in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. Most importantly, the ADC was extremely stable in circulation as shown in pharmacokinetic studies in rodents, demonstrating an exposure profile similar to the unmodified trastuzumab parent antibody. In efficacy studies using an established NCI-N87 colon cancer model (therapeutic setting), a single injection of the Araris Topo 1 ADC at DAR2 at a dose of 52ug/kg (adjusted payload dose) induced superior anti-tumor activity compared to Trastuzumab deruxtecan at DAR of 8, injected at the same payload dose. Complete tumor regression of all tumors (7/7) was obtained at 104ug/kg payload dose and lasted throughout the whole study duration (total 80 days) and was very well tolerated. The data show that Araris Topo 1 ADCs assembled using novel peptide linkers, even at a DAR of as low as 2 have a very efficient anti-tumor activity suggesting optimal drug exposure, targeting and release of the payload. In summary, we show that the Araris Topo1 linker-payloads result in highly potent ADCs with very favorable biophysical properties and extremely efficient payload release as well as an antibody-like exposure profile making them ideal linker-payloads for solid tumor targeting. We anticipate the low-drug load to be favorable in avoiding excessive toxicities in non-targeted tissues. Finally, the Araris bioconjugation technology allows for the generation of tailor-made ADC candidates with improved therapeutic indices. Citation Format: Isabella Attinger-Toller, Rachael Fay, Romain Bertrand, Philipp Probst, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Inducing significant and efficient tumor growth inhibition vs trastuzumab deruxtecan with low drug-load topoisomerase 1 inhibitor ADC using novel peptide linkers for payload conjugation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB219.

The Araris’ site-specific and 1-step linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC with this technology showing a higher therapeutic index compared to polatuzumab-vedotin in preclinical models. Our ADC may represent a safe and efficacious alternative for the treatment of patients with diffuse-large B-cell lymphoma (DLBCL).Using native polatuzumab (non-engineered, same antibody sequence as present in approved polatuzumab-vedotin) as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24hours highly homogeneous and pure ADCs with a well-defined drug-to-antibody ratio (DAR) of 1.9, with a &gt 98% monomer content. The ADC is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In in-vitro assays our ADC demonstrated potent cytotoxicity in four tested cell-lines, similar to the approved polatuzumab-vedotin (Polivy®). Moreover, our anti-CD79b ADC (ARADC) is highly stable in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage. Though highly stable, the ADC is still efficiently released by lysosomal human Cathepsin B cleavage or human liver-lysosome enzymes. Most importantly, the resulting ADC is extremely stable in circulation as shown in pharmacokinetic studies in mice and rats demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab antibody and twice as long as the approved polatuzumab-vedotin (half-life 10d vs 5d).Most importantly, the in vivo efficacy of ARADC (DAR 1.9) was compared with approved polatuzumab-vedotin (DAR 3.5) in two CD79b-expressing tumor models: Granta-519 and Ramos. ARADC provided equal tumor growth inhibition and survival at about half the payload dose relative to polatuzumab-vedotin in both models. At approximately equal payload doses, ARADC treatment led to greater antitumor effects and a considerably longer survival advantage over polatuzumab-vedotin in both models. Finally, the highest non-severely toxic dose (HNSTD) of ARADC was determined at 30mg/kg in a 4-week repeat dose toxicology study in rats. This observation, together with the high anti-tumor potency at low dose - the minimal effective dose (MED), results in an overall 4-6-fold increased therapeutic index (TI).These encouraging results obtained so far indicate that ARADC a) has very favorable biophysical properties, b) shows a clearly defined drug-to-antibody ratio, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) showed an improvement in TI by a factor of 4-6 and ultimately warrant further development of ARADC. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Ramona Stark, Roger Santimaria, Emma Renard, Rachael Fay, Dragan Grabulovski, Bernd Schlereth, Philipp René Spycher. A CD79b targeting ADC with superior anti-tumor activity and therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2910.

The Araris’ site-specific and one-step linker conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-Nectin-4 ADC that shows superior activity to enfortumab-vedotin (Padcev®, EV) in head-to-head in vitro and in vivo studies. Based on enfortumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24 hours highly homogeneous and pure ADCs with a drug-to antibody-ratio (DAR) of approximately 2 and above 98% monomeric content. In in vitro assays on target positive cell-lines, the Araris ADC demonstrated potent cell-cytotoxicity in the low nM-range similar to the approved enfortumab-vedotin which has a DAR of 4. Moreover, our ADC showed excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while EV showed significant payload deconjugation during the 14d incubation period. Interestingly, despite the improved stability, the kinetics for MMAE release was comparable to EV in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. Most importantly, the ADC was extremely stable in circulation as shown in pharmacokinetic studies in rodents, demonstrating an exposure profile comparable to the unmodified enfortumab parent antibody. There was no loss of payload during the 3 week study duration and the calculated half-life of the Araris ADC was approximately 10 days, which is more than 2-fold the half-life of EV. In efficacy studies using a SUM-190PT breast cancer model, a single injection at a dose of 10µg/kg normalized payload induced a complete tumor regression that lasted throughout the whole study duration of more than 100 days. EV administered at the same payload dose showed only a short and transient tumor regression with no animal (0/6) reaching a complete response with some tumor growth retardation until day 20. Our data impressively show that the Araris ADC has superior efficacy even at least 3x lower payload dose compared to EV which are normally needed to achieve complete tumor remission. The very high efficacy even at low dose levels combined with the high plasma stability offer the opportunity to develop an ADC having potentially lower dose-limiting toxicities such as peripheral neuropathy, skin toxicity or neutropenia. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Spycher. Inducing complete and long-lasting tumor eradications at safe and well tolerated doses of a nectin-4 ADC generated with novel peptide linkers for payload conjugation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB174.

Araris' site-specific conjugation technology is improving the safety and efficacy of ADCs without antibody engineering. Our technology is using transglutaminase for site-specific enzymatic payload conjugation to ‘off-the-shelf' antibodies, i.e., without the need to reduce or engineer the antibody. The functionalization takes place site-specifically and stoichiometrically (drug-to-antibody ratio, DAR = 2) at the glutamine residue Q295 in the Fc-part of antibodies. The resulting ADCs show favorable biophysical properties such as high solubility and stability. Additionally, initial data show that linker conjugation does not interfere with Fc-gamma receptor and FcRn binding. Using native polatuzumab (non-engineered, same antibody sequence as present in approved polatuzumab-vedotin) as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24 hours highly homogeneous and pure ADCs with a well-defined DAR of 1.9 (LC-MS). Size exclusion chromatography analysis showed a highly monomeric ADC product (> 96% monomer content) that remained stable under stressed conditions at elevated temperatures. In in-vitro assays our ADCs demonstrated potent cytotoxicity in four tested cell-lines, similar to the approved polatuzumab-vedotin (Polivy®). Moreover, our anti-CD79b ADC (termed ARC-01) is highly stable in mouse, cynomolgus and human sera and is resistant to mouse Ces1c cleavage but is cleaved by the lysosomal human Cathepsin B. Most importantly, the resulting ADC is extremely stable as shown in pharmacokinetic studies in mice demonstrating a very long exposure comparable to the unmodified polatuzumab antibody and twice as long as the approved polatuzumab-vedotin (half-life 10d vs 5d). Most importantly, the in vivo efficacy of our ARC-01 (DAR 1.9) as compared with approved polatuzumab-vedotin (DAR 3.5) was assessed in the Granta-519 tumor model. Animals received a single injection of 0.53 or 2.1 mg/kg polatuzumab-vedotin (Polivy®) and either 0.53, 1 or 2.1 mg/kg of ARC-01. ARC-01 provided equal tumor growth inhibition and survival at about half the payload dose relative to polatuzumab-vedotin (comparison of the 2mg/kg doses and 0.53 mg/kg doses). At an approximately equal payload dose, ARC-01 treatment led to a greater antitumor efficacy and a considerable survival advantage with 6/8 complete tumor remissions over polatuzumab-vedotin with 0/8 complete tumor remission (comparison of the 0.53mg/kg dose of polatuzumab-vedotin and 1mg/kg dose of ARC-01). These encouraging results obtained so far indicate that our linker technology a) allows for fast (< 24 hours) and straightforward manufacturing of ADCs without protein engineering efforts, b) results in ADCs with favorable biophysical properties and a clear defined drug-to-antibody ratio, and c) enables the generation of highly potent and stable, thus safer, next-generation ADCs. Citation Format: Philipp René Spycher, Philipp Probst, Romain Bertrand, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Isabella Attinger-Toller. CD79b targeting ADC with superior pharmacokinetic profile and anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1842.

Biologic treatment options such as tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of inflammatory diseases, including rheumatoid arthritis. Recent data suggest, however, that full and long-lasting responses to TNF inhibitors are limited because of the activation of the pro-inflammatory TH17/interleukin (IL)-17 pathway in patients. Therefore, dual TNF/IL-17A inhibition is an attractive avenue to achieve superior efficacy levels in such diseases. Based on the marketed anti-TNF antibody adalimumab, we generated the bispecific TNF/IL-17A-binding FynomAb COVA322. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. COVA322 was characterized in detail and showed a remarkable ability to inhibit TNF and IL-17A in vitro and in vivo. Through its unique mode-of-action of inhibiting simultaneously TNF and the IL-17A homodimer, COVA322 represents a promising drug candidate for the treatment of inflammatory diseases. COVA322 is currently being tested in a Phase 1b/2a study in psoriasis ( ClinicalTrials.gov Identifier: NCT02243787).

Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2-targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. On the basis of the approved antibody pertuzumab, we have created a panel of bispecific FynomAbs, which target two epitopes on HER2. FynomAbs are fusion proteins of an antibody and a Fyn SH3–derived binding protein. One bispecific FynomAb, COVA208, was characterized in detail and showed a remarkable ability to induce rapid HER2 internalization and apoptosis in vitro. Moreover, it elicited a strong inhibition of downstream HER2 signaling by reducing HER2, HER3, and EGFR levels in vitro and in vivo. Importantly, COVA208 demonstrated superior activity in four different xenograft models as compared with the approved antibodies trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets. Mol Cancer Ther; 13(8); 2030–9. ©2014 AACR.

We introduce a novel and versatile ADC-linker technology that is based on site-specific enzymatic payload conjugation to ‘off-the-shelf’ antibodies, i.e., without the need to reduce or engineer the antibody. The functionalization takes place site-specifically and stoichiometrically (drug-to-antibody ratio, DAR = 2.0) at the Fc-part of antibodies. The resulting ADCs show favorable biophysical properties such as high solubility and stability using different payloads. Additionally, initial ELISA findings show that our modification does not interfere with Fc-gamma receptor and FcRn binding. Importantly, our ADCs showed superior efficacy in different tumor animal models as compared to control Thiomab™ ADCs. Using native trastuzumab (non-engineered) as the targeting antibody and amanitin as payload, we generated within 36 hours highly homogeneous and pure ADCs with a well-defined DAR of 2.0 as confirmed by LC-MS. In in-vitro assays our ADCs demonstrated potent cytotoxicity in all tested cell-lines (SKBR-3, BT-474, JIMT-1, and NCI-N87) as compared to the control Thiomab™ ADCs, most strikingly for the JIMT-1 cell-line: EC50 of 0.15nM vs 2.5nM. In the mouse JIMT-1 tumor model, our anti-HER2 ADC was highly potent and resulted in complete tumor remission in all mice (10/10 mice) at a single dose of 2mg/kg. In contrast, the control Thiomab™-functionalized ADC showed tumor regrowth in 4 out of 10 animals, starting on day 50. The same high potency was observed for the NCI-N87 xenograft model at a dose of 3mg/kg in which tumor growth inhibition was significantly delayed versus the reference ADC (8/10 vs 4/10 animals alive on day 110). These encouraging results obtained so far indicate that our linker technology a) allows for fast (< 36 hours) and straightforward manufacturing of ADCs using different payloads without protein engineering efforts, b) results in ADCs with favorable biophysical properties and a clear defined drug-to-antibody ratio, and c) enables the generation of highly potent and stable, thus safer, next-generation ADCs. Citation Format: Philipp Rene Spycher, Julia Carina Frei, Jöri Elias Wehrmüller, Isabella Attinger-toller, Dragan Grabulovski, Torsten Hechler, Michael Kulke, Andreas Pahl, Martin Behe, Roger Schibli. Overcoming limitations of current Antibody-Drug Conjugates (ADCs) by a novel linker technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-106.

Acute myeloid leukemia (AML) is an aggressive blood cancer with neoplastic infiltration of leukemic blasts in blood, bone marrow and viscera. Current treatment options have limited efficacy, and the 5-year survival rate is only 27%. CD3 bispecific antibodies that re-direct T cells towards the tumor have shown promising efficacy in hematological malignancies. The CD3/CD19 bispecific antibody blinatumomab has recently been approved by the FDA for the treatment of B-cell acute lymphocytic leukemia (ALL) (Kantarjian H et al (2017) NEJM). We evaluated COVA4231, a CD3/CD33 bispecific FynomAb, preclinically as therapeutic candidate for AML. CD33 is a cell surface receptor expressed on blasts of the majority of AML patients (Walter RB (2014) Expert Opin Ther Targets). COVA4231 was constructed by fusing the CD33-specific Fynomer D5 - a small (7 kDa) globular protein derived from the human Fyn SH3 domain with engineered affinity for CD33 - to a CD3-specific antibody with a novel silent IgG1 Fc. COVA4231 induced T cell activation and cytokine release in a CD33-dependent manner, and elicited potent T cell mediated cytotoxicity of CD33-expressing target cells in vitro with EC50 in the range of 4 - 29 pM (E:T cell ratio of 2:1, KG-1 or MOLM-13 as target cells). COVA4231 showed potent activity against 13 out of 15 primary AML blast samples ex vivo at E:T cell ratio of 1:1. The in vivo efficacy of COVA4231 was investigated in a subcutaneous HL-60 leukemia xenograft model in NSG mice in the presence of human T cells. COVA4231 was highly active across a wide dose range (0.05 - 5 mg/kg) and prevented tumor outgrowth in all treated mice. COVA4231 demonstrated an IgG1-like pharmacokinetic profile in mice with a terminal half-life 15.8 days (total drug), providing the opportunity to avoid continuous intravenous infusion protocols required for established CD3 bispecific formats (e.g. (scFv)2). In conclusion, COVA4231 is a highly active therapeutic candidate in vitro, in vivo and ex vivo, has IgG-like pharmacokinetics and is a promising therapeutic candidate for further preclinical and clinical development. Citation Format: Kristina Klupsch, Vanessa Baeriswyl, Roland Scholz, Joana Dannenberg, Roger Santimaria, David Senn, Elena Kage, Adrian Zumsteg, Isabella Attinger-Toller, Ulrike von der Bey, Susann König-Friedrich, Fanny Dupuy, Wibke Lembke, Clara Albani, Severin Wendelspiess, Lucijana Dinkel, Chelsea J. Gudgeon, Roland B. Walter, Julian Bertschinger, Simon Brack. COVA4231, a potent CD3/CD33 bispecific FynomAb with IgG-like pharmacokinetics for the treatment of acute myeloid leukemia [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 1787.

CD3 bispecific therapies retargeting T cells to tumors have recently demonstrated striking activity in patients. Several CD3 bispecific antibodies directed against various tumor targets are currently being investigated in the clinic across different tumors. One limitation of these therapies is the risk of target-related toxicity due to low-level expression of tumor antigen in normal tissue. In this study we have engineered a bispecific CD3/HER2 FynomAb, COVA420, which redirects T cells with high potency and selectivity to tumor cells with high HER2 expression in vitro and in vivo. COVA420 activity depends on high HER2 density as no activity was observed on cells with lower HER2 levels as found in human normal tissue. These results suggest that COVA420 may spare normal tissue expressing low levels of HER2 while still having uncompromised efficacy on tumor cells with high HER2 expression. This concept may be applied to other cancer antigens that otherwise cannot be targeted by T cell redirecting approaches, and may therefore expand the applicability of CD3 bispecific FynomAbs to a larger number of solid tumors.

Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2 targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. Fynomers are small 7 kDa globular proteins derived from the SH3 domain of the human Fyn kinase (Fyn SH3) that can be engineered to bind with antibody-like affinity and specificity to virtually any target of choice. Fynomers can be fused to N-terminal and/or C-terminal ends of antibodies to generate multispecific therapeutics (FynomAbs) with tailored architectures. FynomAbs can be produced using standard antibody technology (GMP production yield of 3.3 g/L at 1000 L scale achieved), and show IgG-like biophysical properties and pharmacokinetic profiles. Based on the approved antibody pertuzumab we have created a panel of bispecific FynomAbs which target two epitopes on HER2. The activity of the HER2 targeting FynomAbs was found to depend on the FynomAb architecture, i.e. the spatial arrangement of the binding sites of antibody and the Fynomer. The most potent of these FynomAbs, termed COVA208, demonstrated superior tumor cell growth inhibition in vitro compared to pertuzumab and trastuzumab. COVA208 was characterized in detail and showed an increased ability to induce rapid HER2-internalization and apoptosis in vitro. Moreover, it elicited a stronger inhibition of downstream HER2 signaling which was accompanied by a reduction of HER2, HER3 and EGFR levels in vitro and in vivo. The therapeutic potential of COVA208 has been demonstrated in vivo in four different HER2 mouse models, where COVA208 exhibited excellent anti-tumor activity. Importantly, COVA208 demonstrated superior activity in vivo compared to trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets. Citation Format: Babette Schade, Simon Brack, Isabella Attinger-Toller, Kristina Klupsch, Richard Woods, Helen Hachemi, Ulrike von der Bey, Susann Koönig-Friedrich, Julian Bertschinger, Dragan Grabulovski. A bispecific HER2 targeting FynomAb with superior anti-tumor activity and novel mode of action. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 658. doi:10.1158/1538-7445.AM2014-658

There is increasing evidence that T cells are able to control tumor growth and increase survival of cancer patients. However, tumor-specific T cell responses are difficult to mount and sustain in cancer patients, and are limited by numerous immune escape mechanisms of tumor cells. A promising approach in the immunotherapy of cancer is to engage T cells to target tumor cells using bispecific therapeutics targeting a tumor-associated surface antigen and CD3e on T cells. Such therapeutics elicit T cell mediated lysis of tumor cells independent of T cell specificity. Fynomers are small 7 kDa globular proteins derived from the SH3 domain of the human Fyn kinase (Fyn SH3) that can be engineered to bind with antibody-like affinity and specificity to virtually any target of choice. Fynomers can be fused to N-terminal and/or C-terminal ends of antibodies to generate multispecific therapeutics (FynomAbs) with tailored architectures. FynomAbs can be produced using standard antibody technology (GMP production yield of 3.3 g/L at 1000 L scale achieved), and show IgG-like biophysical properties and pharmacokinetic profiles. We have generated a novel bispecific FynomAb which can simultaneously bind HER2 on tumor cells and CD3 on T cells. The bispecific HER2/CD3 targeting FynomAb COVA420 potently redirected T cells to HER2 expressing tumor cells showing picomolar tumor cell lysis activity. We present for the first time that a tailored architecture of the FynomAb leads to optimal tumor cell killing properties. The activity of COVA420 was found to be highly specific, as no lysis of cells was observed in the absence of HER2 expression. In addition, COVA420 demonstrated an antibody-like pharmacokinetic profile in mice. We anticipate that the increased half-life of T cell recruiting FynomAbs compared to other bispecific formats translates into a significant benefit for patients, since it circumvents the need for continuous infusion and prolongs the intervals between successive treatments. In summary, bispecific T cell recruiting FynomAbs represent a novel platform technology to redirect T cells to tumor cells with optimal biophysical properties, long-half lives and tailored architectures. Citation Format: Ulrich Wuellner, Fabian Buller, Kristina Klupsch, Simon Brack, Irene Zbinden, Roger Santimaria, Isabella Attinger-Toller, Susann König-Friedrich, Julian Bertschinger, Dragan Grabulovski. A bispecific HER2/CD3 targeting FynomAb with excellent tumor killing and favorable pharmacokinetic properties. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2014-656

3066 Background: A promising approach in the immunotherapy of cancer is to recruit T cells to attack tumor cells using bispecific therapeutics targeting a tumor-associated surface antigen and CD3 o...