Antibody Technology
#50 HTI-1511, a novel anti-EGFR-ADC, overcomes mutation resistance and demonstrates signifıcant activity against multiple tumor types in pre-clinical studies.Jesse D. Bahn,1Feng Gao,1Lei Huang,1Barbara Blouw,1 Chun-mei Zhao,1Kelly Chen,1Susan Zimmerman,1Erin K. Wise,2Maria L. Mancini,2 Matthew Bird,3William McDowell,3Curtis B. Thompson,1Sanna Rosengren,1 Christopher D. Thanos1.1Halozyme Therapeutics, San Diego, CA;2Champions Oncology, Inc, Baltimore, MD;3Abzena, Antitope Limited & PolyTherics Limited, Cambridge, United Kingdom.
Multiple solid tumor types over-express epidermal growth factor receptor (EGFR). Antibodies that target the receptor are often accompanied by adverse skin reactions due to interaction with receptors expressed in normal tissue. Additionally, downstream mutations (KRAS, BRAF) within tumors can result in EGFR-indepen-dent activation and resistance to treatment. We have previously described HTI-1511, an antibody-drug conjugate in pre-clinical development that targets EGFR.
HTI-1511 carries the potent cytotoxin MMAE and a novel bis-alkylating linker, connected to a monoclonal antibody engineered to have improved specifıcity for EGFR in the tumor microenvironment (Huang et. al. AACR National Meeting, 2016, New Orleans, LA). Here we screened a panel of over 70 tumor cell lines derived from various solid tumor malignancies for both EGFR expression by flow cytometry and sensitivity to cell growth inhibition by HTI-1511 in vitro. Cell lines derived from head and neck squamous cell carcinoma (SCC15, CAL27, FaDu, CAL33, SCC25 [IC500.52 nM - 3.1 nM]), non-small cell lung cancer (HCC827, NCI-H1666, PC-9, NCI-H1650 [IC500.04 nM - 6.2 nM]), and pancreatic carcinoma (BxPC-3, PANC-1, AsPC-1 [IC500.99 nM - 4.44 nM]) showed particular sensitivity to HTI-1511. In conjunction, HTI-1511 effıcacy was assessed in vivo for tumor growth inhibition (TGI) in several human tumor xenograft models. Evaluations in the human xeno-grafts A431 (epidermoid, 93% TGI at 3.0 mg/kg, p⬍0.05), BxPC3 (pancreatic,
⬎100% TGI at 3.0 mg/kg, p⬍0.05), AsPC-1 (pancreatic, ⬎100% TGI at 3.0 mg/kg, p⬍0.05), and FaDu (HNSCC, ⬎100% TGI at 3.0 mg/kg, p⬍0.05) indicated dose dependent tumor regressions in all cases, and no observed toxicity when adminis-tered weekly at dose levels up to 3 mg/kg for up to eight weeks. HTI-1511 anti-tumor activity was also investigated in a group of patient derived xenograft (PDX) models.
An initial study evaluated weekly administration of a single dose level of 2.5 mg/kg HTI-1511 in three different PDX models. TGI of 83% (p⬍0.05) was observed in a BRAFmutcolorectal cancer model and 57% (p⬍0.05) in a wildtype colorectal cancer model, as well as 46% (not signifıcant) TGI seen in a wild type renal cell carcinoma model. A separate study in a NSCLC (KRASmut) PDX model demonstrated a dose dependent response with greater than 100% TGI at 1.0 and 3.0 mg/kg (both p⬍0.05, and also p⬍0.05 to a non-cognate antibody ADC control). The non-cognate anti-body control yielded 67.4% (not signifıcant) TGI by itself compared to the vehicle control group. These results support further development of HTI-1511 as a possible treatment for EGFR overexpressing tumors, including those with downstream acti-vating mutations in the KRAS/BRAF pathway.
#51 Characterization of the mechanism of action, pharmacodynamics and preclinical safety of ADCT-402, a pyrrolobenzodiazepine (PBD) dimer-con-taining antibody-drug conjugate (ADC) targeting CD19-expressing hemato-logical malignancies.Francesca Zammarchi,1Simon Corbett,2Karin Have-nith,1Narinder Janghra,2Konstantinos Kiakos,2Teresa Marafıoti,2David G.
Williams,3Simon Chivers,1Phil W. Howard,3John A. Hartley,2Patrick H. van Berkel1.1ADC Therapeutics, London, United Kingdom;2University College Lon-don, LonLon-don, United Kingdom;3Spirogen/MedImmune, London, United King-dom.
ADCT-402, currently in Phase I clinical trials for B-cell hematological malig-nancies, is an ADC composed of a recombinant humanized IgG1 against human CD19, stochastically conjugated via a cleavable linker to a PBD dimer cytotoxin (DAR of 2.3). PBD dimers, DNA minor groove interstrand cross-linking agents, are gaining increasing attention and are currently being tested as the ADC war-heads in several clinical trials. ADCT-402 has potent and targeted cytotoxicity against a panel of human lymphoma and leukemia cell lines in vitro. In vivo, ADCT-402 demonstrates dose-dependent antitumor activity against Burkitt’s lymphoma xenograft models. Moreover, ADCT-402 is markedly superior to maytansinoid- and auristatin-based CD19-targeting ADCs in the Ramos xeno-graft model. In a rat toxicology study, a single dose of ADCT-402 at 2 mg/kg is well tolerated with a favorable PK profıle and excellent stability in vivo. The current study aimed to further defıne the mechanism of action (MOA) of ADCT-402 and validate its pharmacology and preclinical safety for clinical de-velopment. CD19 is a clinically validated target with restricted normal tissue expression and a widespread expression in the majority of B-cell malignancies.
Importantly, we show here the consistent expression of CD19 in matched sam-ples (initial diagnosis and relapsed/refractory) from panels of lymphoma pa-tients, indicating that relapsed/refractory patients are appropriate for treatment with ADCT-402. ADCT-402 was shown to be effıciently internalized by CD19⫹ cells in vitro. Moreover, in line with the PBD dimer MOA, following a 2 hour exposure to ADCT-402, DNA interstrand cross-links reached a peak between 8 - 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of cross-link formation for the PBD dimer warhead alone was observed imme-diately after 2 hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA cross-links. In SCID mice s.c. implanted with Ramos cells, a single dose of ADCT-402 was administered at 0.33 or 1 mg/kg. Twenty-four hours after treatment, excised tumors showed a dose proportional increase in intensity of staining by an anti-PBD payload antibody, as well as in DNA cross-linking and in␥-H2AX formation. In contrast, no DNA cross-linking was ob-served in matched lymphocyte samples. The toxicity of ADCT-402 was further evaluated in a repeat dose cynomolgus monkey study. ADCT-402 was clinically well tolerated with an acceptable off-target safety profıle. The PK of the ADC was consistent with normal antibody clearance with a half-life of about 12 to 17 days.
These data confırm the MOA of ADCT-402 and provide relevant pharmacody-namic assays and preclinical safety assessment to guide the clinical development of this promising ADC in B-cell malignancies.
#52 Mechanistic and benchmarking studies of ADCT-502, a pyrroloben-zodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) tar-geting HER2-expressing solid tumors.Francesca Zammarchi,1Halla W. Rein-ert,2 Narinder Janghra,2 Simon Corbett,2 Maria Mellinas-Gomez,3 Sajidah Chowdhury,3Neha Arora,3Peter Tyrer,3Francois Bertelli,3David G. Williams,3 Philip W. Howard,3John A. Hartley,2Patrick H. van Berkel1.1ADC Therapeu-tics, London, United Kingdom;2University College London, London, United Kingdom;3Spirogen/MedImmune, London, United Kingdom.
ADCT-502 is an ADC composed of an engineered version of humanized IgG1 trastuzumab, directed against human HER2, site-specifıcally conjugated to the highly cytotoxic PBD-based linker-drug tesirine (drug-antibody ratio of 1.7). In vitro, ADCT-502 has highly potent and targeted cytotoxicity against various solid cancer cell lines. In vivo, ADCT-502 demonstrates strong and durable antitumor activity in mouse xenografts with various levels of HER2, but is inac-tive in a HER2-negainac-tive xenograft. ADCT-502 is stable, well tolerated and has a favorable PK profıle both in rat and cynomolgus monkey. The current study aimed to defıne further the mechanism of action of ADCT-502 and to bench-mark its activity in xenograft models against ado-trastuzumab emtansine (T-DM1), the ADC currently approved for the treatment of HER2⫹ metastatic breast cancers. ADCT-502 bound and internalized effıciently in JIMT-1 cells (HER2⫹) and co-localized with lysosomes within 2 hours. PBD dimers bind in the DNA minor groove and exert cytotoxicity via the formation of DNA strand cross-links. Following a 2-hour exposure to ADCT-502, DNA inter-strand cross-linking peaked between 12 and 24 hours, after which cross-links persisted at least 36 hours. In contrast, cross-link formation by an equimolar concentration of warhead alone, peaked immediately following drug exposure and a non-targeted ADC did not produce DNA crosslinks in these cells. More-over, ADCT-502 showed indirect bystander killing activity in HER2-negative MDA-MB-468 cells incubated with conditioned medium from ADCT-502-treated HER2⫹ SK-BR-3 cells. In vivo, antitumor activity of ADCT-502 was compared to T-DM1 in both cell line- and patient-derived-xenograft (PDX) models. For example, in a HER2 1⫹, FISH- breast cancer PDX, ADCT-502 showed dose-dependent antitumor activity resulting in 1/8 and 8/8 TFS after a single dose at 0.1 and 0.2 mg/kg, respectively. Conversely, a single dose of T-DM1 at 30 mg/kg showed only marginal activity compared to the control.
EXPERIMENTAL AND MOLECULAR THERAPEUTICS: Antibodies, Bispecifıcs, and Antibody-Drug Conjugates
Similarly, in a HER2 1⫹, FISH- esophageal cancer PDX, while a single dose of ADCT-502 at 0.44 mg/kg resulted in strong and durable antitumor activity, single doses of T-DM1 at either 10 or 30 mg/kg showed no activity compared to the control. These data confırm that the mechanism of cell killing of ADCT-502 is via target-specifıc internalization and subsequent cross-linking of DNA. They also show superior in vivo antitumor activity of ADCT-502 compared to T-DM1 in various tumor xenografts, including those with low HER2 levels. Taken to-gether, these results support the development of ADCT-502 not only in patients that have become resistant/refractory to T-DM1, but also in patients whose tumors express low levels of HER2, and are not eligible for treatment with T-DM1.
#53 Antibody-drug conjugates (ADCs) of peptide-linked Indolino-Benzo-diazepine (IGN) DNA-alkylator provides improved anti-tumor activity over that of a crosslinker.Michael L. Miller, Manami Shizuka, Jose F. Ponte, Leanne Lanieri, Dilrukshi Vitharana, Qifeng Qiu, Emily E. Reid, Katie E. Archer, Rui Wu, Erin K. Maloney, Olga Ab, Jan Pinkas, Ravi V. Chari. ImmunoGen, Inc., Waltham, MA.
We recently disclosed highly active antibody-drug conjugates (ADCs) that incorporated the novel DNA alkylating indolino-benzodiazepine (termed IGN) dimer, DGN549 (IGN-P1). The stereochemistry of the alanyl moiety of the protease-cleavable alanine-alanine linker used was shown to impact ADC catab-olism, bystander killing activity, and in vivo effıcacy (Shizuka, et al., AACR 2016
#2959). Building upon these results, here we describe preclinical results from a head-to-head comparison of ADCs of the mono-imine containing DGN549 with its corresponding DNA cross-linking diimine version, IGN-P1 diimine.
IGN-P1 diimine and DGN549 were conjugated to a folate receptor-␣ (FR␣)-binding antibody and an EpCAM-(FR␣)-binding antibody. The resulting ADCs dem-onstrated similar high in vitro potency (IC50⬃3-100 pM) and specifıcity to-wards several cancer cell lines. Further in vitro studies revealed that the DNA alkylating anti-FR␣-DGN549 ADC demonstrated superior bystander cell-kill-ing activity compared to its DNA crosslinkcell-kill-ing counterpart, anti-FR␣-IGN-P1 diimine. In vivo, this improved bystander killing ability translated into better in vivo activity for the DNA alkylating ADC. In an endometrial tumor xenograft model established with Ishikawa cells, the anti-FR␣-DGN549 induced complete regressions at a single dose of 140g/kg Ab dose (equivalent to 5 g/kg linked IGN). The cross-linking anti-FR␣-IGN-P1 diimine had to be used at twice the dose to achieve the same level of anti-tumor activity. The in vivo tolerability in CD-1 mice also displayed differences in the two ADCs. We found that the ADC of the DNA crosslinker was at least two-fold less tolerated than the correspond-ing ADC of the DNA alkylator. These results indicate that a⬃4 fold greater therapeutic index can be achieved when using a DNA alkylating mono-imine DGN549 ADC as compared to the DNA crosslinking IGN-P1 diimine ADC.
#54 The many faces of antibody in G protein-coupled receptor biology.Lei Chen, Wing-Tai Cheung. School of Biomedical Sciences, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong.
G protein-coupled receptors (GPCRs), the largest family of cell-surface recep-tor proteins mediating signal transmission, play a pivotal role in many physio-logical functions and are involved in multiple diseases. Although receptor activ-ities of GPCRs are successfully modulated by many small molecules which represent 30% of all marketed drugs today, in fact only a few GPCR members are targeted and many more GPCRs of interest are intractable targets of small mol-ecules, like orphan GPCRs and GPCRs with large binding sites. Besides, small molecule drugs rarely trigger GPCR-mediated apoptosis or induce direct cell killing, which is crucial for cancer therapies. Therefore, for these GPCRs, anti-body-based drug would be a good alternative. Despite being eagerly sought for, the production of monoclonal antibody (mAb) targeting GPCR is hindered by the low expression of recombinant GPCR on cell surface, and relatively small exposed regions with glycosylation and conformational heterogeneity. There-fore those conventional methods that were well established for generation of mAbs targeting soluble proteins usually showed frustrating results in the case of GPCRs. To bypass the production and purifıcation of recombinant GPCR pro-teins, we developed a novel approach of transplanting immunogenic conforma-tional epitopes of GPCR into an antibody scaffold to make a water soluble sur-rogate antigen of GPCR, named as GPCR-antigenized antibody which can be easily and abundantly produced in bacteria for animal immunization. In cou-pled with phage display technology and cell panning strategy, which allow the retrieval of specifıc binders from a huge number of candidates, we have success-fully isolated scFv antibodies for two human GPCRs, the MAS1 receptor and the chemokine receptor CXCR4. These antibodies showed good specifıcity in im-munofluorescent staining, flow cytometry analysis, western protein immuno-blot and immunoprecipitation assay. The results demonstrated this novel
ap-proach may offer a generic and effective method to generate specifıc monoclonal antibody targeting GPCR for diagnostic and therapeutic applications. [The proj-ect is partly funded by a CUHK dirproj-ect grant (4054300)]
#55 Generation of half-life extended anti-CD33 BiTE® antibody constructs compatible with once-weekly dosing.Tara L. Arvedson,1Mercedesz Balazs,2 Pamela Bogner,3Kurt Black,1Kevin Graham,1Anja Henn,3Matthias Friedrich,3 Patrick Hoffmann,3Roman Kischel,3Peter Kufer,3Ralf Lutterbuese,3Markus Muenz,3Tobias Raum,3Benno Rattel,3Karen Rex,1Dan Rock,2Oliver Thomas,3 Joachim Wahl,3Andreas Wolf,3Angela Coxon1.1Amgen, Thousand Oaks, CA;
2Amgen, San Francisco, CA;3Amgen, Munich, Germany.
T cell engaging bispecifıc antibody constructs (BiTE®), such as blinatumomab which targets CD19-positive cells, have shown great promise for treating certain CD19-positive hematological malignancies. Blinatumomab comprises a single chain Fv (scFv) that binds CD19 and a scFv that targets the T cell CD3 protein.
The molecular weight of this “canonical” BiTE® is⬃ 55 kDa, making it suscep-tible to kidney-mediated clearance and resulting in a short serum half-life (⬃ 4 hours). To maintain effective serum concentrations, canonical BiTE® antibody constructs must be administered by continuous IV (cIV) infusion. While there are many advantages associated with cIV administration (e.g., safety and uni-form PK profıle), patient convenience could be enhanced if the BiTE® antibody construct were compatible with once-weekly administration. To achieve this, the serum half-life of the BiTE® antibody construct would need to be extended. A canonical BiTE® targeting CD33 (AMG 330) is currently being evaluated in a phase I clinical trial. Like blinatumomab, AMG 330 is dosed cIV. To extend the serum half-life of AMG 330 and enable once-weekly dosing, several approaches were evaluated including fusion of AMG 330 to human albumin and Fc-con-taining moieties. Each of these half-life extended (HLE) constructs was evalu-ated in vitro, in mouse xenograft models and in non-human primates. In vitro assays evaluated 1) binding to both human and cynomolgus CD33 and CD3 proteins, and 2) cytotoxicity using human and cynomolgus target and effector cells. In each of these assays the canonical and HLE BiTE® antibody constructs demonstrated similar activity: single-digit nM binding and single digit pM cy-totoxicity. Canonical and HLE BiTE® antibody constructs were subsequently evaluated in an orthotopic mouse model in which MOLM13 cells were admin-istered IV and activated human T cells were adminadmin-istered IP two days later. The Fc-based HLE BiTE® antibody constructs provided a similar survival advantage when administered Q4D or Q5D as the canonical BiTE® when administered QD.
However, the albumin fusion– based HLE BiTE® was less effıcacious when ad-ministered Q4D than the QD- adad-ministered canonical BiTE®. Lastly, the PK/PD relationship was evaluated for each of the constructs in non-human primates.
The serum half-lives varied from 6 hours for the canonical BiTE® to 44-167 hours for the HLE BiTE® antibody constructs. Each of the HLE BiTE® antibody con-structs showed on-target depletion of CD33-positive monocytes and neutro-phils in the blood and depletion of CD33-positive cells in the bone marrow.
These data demonstrate that half-life extended BiTE® antibody constructs can be generated that retain comparable in vitro and in vivo activity as a canonical BiTE® and achieve a serum half-life compatible with once weekly dosing.
#56 Antibody-drug conjugates containing glucuronide-tubulysin pay-loads display activity in MDRⴙ and heterogeneous tumor models. Patrick J. Burke, Joseph Z. Hamilton, Joshua H. Hunter, Julia H. Cochran, Thomas A.
Pires, Christopher I. Leiske, Kim K. Emmerton, Peter D. Senter, Robert P. Lyon, Scott C. Jeffrey. Seattle Genetics, Inc., Bothell, WA.
While antibody-drug conjugates (ADCs) fınd increasing application in can-cer treatment regimens, de novo or treatment-emergent resistance mechanisms could impair clinical benefıt. Two resistance mechanisms that emerge under continuous ADC exposure in vitro include upregulation of transporters that confer multidrug resistance (MDR⫹) and loss of cognate antigen expression.
New technologies that circumvent these resistance mechanisms may serve to extend the utility of next generation ADCs. Recently, we developed the quater-nary ammonium linker system to expand the scope of conjugatable payloads to include tertiary amine-containing compounds and applied the linker to tubu-lysins, a highly potent class of microtubule disrupting agents that maintain ac-tivity in MDR⫹ cell lines. Quaternary ammonium-linked glucuronide-tubuly-sin drug-linkers were synthesized and evaluated as ADCs. The resulting conjugates were potent and immunologically specifıc across a panel of cancer cell lines, including those displaying the MDR phenotype. The ADCs also dem-onstrate potent bystander activity in a co-culture model containing a mixture of antigen-positive and -negative cell lines. Incorporation of a PEG12 side chain in the linker enabled loading at 8-drugs/Ab for increased in vivo potency while maintaining suitable ADC pharmacokinetic properties. In vivo, the glucuro-nide-tubulysin conjugates displayed activity in MDR⫹ xenograft models and EXPERIMENTAL AND MOLECULAR THERAPEUTICS: Antibody Technology
bystander activity in an admixed Ag⫹/Ag- heterogeneous tumor model. Thus, the glucuronide-tubulysin drug-linkers represent a promising new payload for ADCs, combining conjugate potency in the presence of the MDR phenotype with robust activity in models of tumor antigen heterogeneity.
#57 Generation and characterization of novel anti-glycan monoclonal an-tibody against acute myeloid leukemia.Chong Kok Teo,1Wee Joo Chng,2 Andre Boon Hwa Choo1.1Bioprocessing Technology Institute, Singapore, Singa-pore;2National University Cancer Institute, Singapore, Singapore, Singapore.
Hematological malignancies encompass a wide spectrum of cancers from hematopoietic and lymphoid tissues. Under this grouping, the various forms of leukemia, lymphoma and myeloma have been classifıed into more than 60 dis-tinct disease types, each having particular clinical features and disease outcomes.
Among these diseases, curative treatments with high long-term survival rate have been developed for some, e.g. Chronic Myeloid Leukemia, while such ef-fective regimens are still unavailable for many others, e.g. Multiple Myeloma and Acute Myeloid Leukemia. In this study, a panel of antibodies was raised against
Among these diseases, curative treatments with high long-term survival rate have been developed for some, e.g. Chronic Myeloid Leukemia, while such ef-fective regimens are still unavailable for many others, e.g. Multiple Myeloma and Acute Myeloid Leukemia. In this study, a panel of antibodies was raised against