services and technologies brochure

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1 services and technologies brochure

2 Foreword Working with world-leading companies and academic institutions across many therapeutic areas, Abzena is proud to offer its suite of complementary services and technologies to enable the development of better biopharmaceuticals in order to improve outcomes for both patients and payers. John Burt Chief Executive Officer Abzena provides a suite of complementary services and technologies to R&D organisations to improve the chances of successful development of therapeutic proteins and antibodies, and it is therefore poised to capitalise on the growth of the biopharmaceutical market. The Group s technologies and services are provided through its wholly owned subsidiaries, PolyTherics and Antitope. PolyTherics and Antitope have been developing and offering their technologies and services for over 10 years and their scientists have provided expertise to a wide range of companies, including most of the top 20 biopharmaceutical companies, many large and small public and private biotech, and academic groups, across the world. 2

3 Antitope provides immunogenicity assessment and protein engineering services to create humanized antibodies and deimmunised proteins. In addition, the company can develop manufacturing cell lines for antibodies and therapeutic proteins as they advance towards clinical development. Antitope s services are tailored for each project according to the client s objectives. Experienced project teams are assigned to each study to deliver quality results in a timely manner. These results inform and enable the further development of our client s products, whether they are a major biopharmaceutical company or an academic institution pursuing translational research programs. Immunogenicity assessment 4 Antibody humanization 6 Protein deimmunisation 7 Cell line development 8 Additional services 9 PolyTherics bioconjugation expertise has led to the development of technologies to produce better antibody drug conjugates (ADCs) and optimisation of the pharmacokinetics and pharmacodynamics of therapeutic proteins, including antibody fragments and novel protein scaffolds. Collaborations with a wide range of partners have successfully delivered product candidates with improved product profiles, including extended in vivo half-life for therapeutic proteins and more stable and homogeneous ADCs. Antibody drug conjugates 10 Optimisation of pharmacokinetics 12 3

4 Immunogenicity assessment Therapeutic antibodies and proteins can induce an immunogenic response in patients, potentially leading to rapid clearance, neutralisation of therapeutic effect and toxicities. CD4+ T cell responses are the primary driver of antibody mediated memory immune responses and the frequency of CD4+ T cell responses to biopharmaceuticals correlates with clinical immunogenicity. Antitope has developed ex vivo technologies that assess CD4+ T cell responses to antibodies and proteins and in silico tools to predict the presence of T cell epitopes. These technologies are used during early development to enable selection of the least immunogenic lead candidate and to identify formulations which have the least impact on potential immunogenicity, thereby reducing the risk of failure in the clinic. EpiScreen TM - ex vivo T cell assays EpiScreen is an accurate and sensitive ex vivo method for immunogenicity assessment of antibodies and other proteins which can provide a guide to the potential immunogenic response that might occur in patients. EpiScreen results correlate with immunogenicity in the clinic EpiScreen T cell assays provide an immunogenicity risk assessment based on the magnitude and frequency of responses to test samples. A clear correlation has been shown between the percentage of T cell responses from EpiScreen time course T cell assays with a range of antibodies and proteins and the frequency of patients who develop anti-drug antibodies. 45 EpiScreen donor response (%) TPO Humira Hirundin Xolair Synagis Avastin Herceptin Enbrel Cantuzumab R 2 = 0.83 IFNbeta MLN02 IFNalpha stnfr1a Infliximab Campath Clinical immunogenicity (%) An analysis of 16 clinical-stage biologics where the frequency of donor responses by EpiScreen analysis demonstrates a high correlation with the average frequency of patients developing anti-drug antibodies reported in clinical studies (Baker et al., 2007 and Barker et al., 2010) A33 >100 number of EpiScreen TM projects completed 4

5 Applications Lead identification Whole protein analysis using the EpiScreen Time Course or DC T Cell assay can differentiate between related protein variants, such as humanized antibody candidates, to enable the selection of leads with the lowest immunogenic potential. These can be benchmarked against proteins of known clinical immunogenicity and facilitate a decision on whether to re-engineer the antibody or protein to reduce the risk. Formulation testing The EpiScreen assay can be used to assess the impact of formulations with different excipients on the potential immunogenicity of the formulated protein or antibody. Although human serum albumin has been widely used, serum free formulation alternatives have been sought to improve product safety with reduced immunogenicity. For example, EpiScreen assays have been used to compare the potential immunogenicity of two formulations of IFN-β, the results of which have been subsequently confirmed in a clinical trial. T cell epitope mapping The EpiScreen epitope mapping assays use synthetic 15mer peptides spanning the protein sequence of interest to provide a highly sensitive method for mapping CD4+ T cell epitopes. These assays detail the magnitude and frequency of response to each peptide and generate a map of epitope locations within the test antibody or protein. Identification of T cell epitopes during the pre-clinical development phase of a protein therapeutic allows for potential design of deimmunised variants with a lower risk of immunogenicity in patients. T cell epitope mapping technology has been applied to the deimmunisation of monoclonal antibodies, enzymes, bacterial proteins, toxins and other non-human proteins. Aggregate analysis The presence of aggregates can lead to enhanced immunogenic responses against therapeutic antibodies and proteins. EpiScreen TM has been used to assess the effects of aggregates on the risk of immunogenicity and thus aid in the development of improved manufacturing processes and the selection of the most suitable formulation for use in clinical trials. 5..We are delighted to be working with Antitope to deimmunise our protein warhead using their proven technology platform.. Kurt Gehlsen Research Corporation Technologies VP & CSO itope and TCED TM - in silico itope and TCED are in silico tools to rapidly screen antibodies and proteins for potential immunogenicity. Combined, they provide a more accurate prediction of T cell epitopes than other in silico technologies that rely solely on MHC class II binding analysis. itope TM itope is a proprietary in silico molecular modelling technology which models the binding between amino acid side chains of a peptide and specific binding pockets within the binding grooves of 34 human MHC class II alleles. TCED TM Data from EpiScreen T cell epitope mapping studies conducted over a decade have enabled us to develop a database of potentially immunogenic helper CD4+ T cell epitopes. Sequences from antibodies and proteins that are candidates for development as therapeutic agents can be analysed for homology to these known T cell epitopes, enabling the rapid and accurate in silico identification of their potential to produce an immunogenic response. itope TM & TCED TM A powerful combination TCED & itope can be combined for rapid analysis of multiple sequences such as those from therapeutic antibody candidates, in order to identify a strong lead sequence with a reduced risk of containing T cell epitopes. For more information [email protected] Immunogenicity Assessment Antibody & Protein Engineering Cell Line Development Antibody Drug Conjugates Optimisation of Pharmacokinetics

6 Antibody and Protein Engineering The development of anti-drug antibodies (ADAs) to therapeutic antibodies and proteins can reduce their efficacy, through their rapid clearance or neutralization and potentially cause toxicity by cross-reacting with other proteins in the body. Antitope s Composite Protein and Composite Human Antibody engineering technologies combine the best attributes of deimmunisation and humanization techniques to create new antibodies and proteins without immunogenic sequences to minimise the risk of an immunogenic response when they enter clinical development. Composite Human Antibodies TM Antibody humanization using Antitope s Composite Human Antibody technology uses multiple sequence segments derived from variable (V) regions of unrelated human antibodies, unlike other technologies that typically use a single human V region framework as acceptors for complementarity determining regions (CDRs) from starting antibodies (typically rodent or phase-display derived). Through careful selection of human sequence segments and the application of in silico tools, CD4+ T cell epitopes can be avoided to reduce the risk of immunogenicity compared to standard humanized antibodies. Composite Human Antibodies are screened against the test antigen to ensure that the affinity and specificity of the antibody is maintained. Advantages of Composite Human Antibodies TM Reduced immunogenicity compared to humanized and fully-human antibodies Excellent retention of affinity and specificity compared to the starting monoclonal antibody Proprietary technology, avoids other humanization patents Confirmation of T cell epitope avoidance by Antitope s EpiScreen technology Stable cell line and sample of lead provided as standard Applications Different starting antibody formats Composite Human Antibodies TM can be generated using a non-human antibody as a reference or previously humanized or fully-human antibodies. Different antibody formats Composite Human Antibodies TM can be constructed with any human constant regions (typically human IgG1 or IgG4 / κ) or generated in a different format including Fab, single-chain Fv, bispecific Fv, antibody-protein fusions. Retention or improvement of function Test antigen can be used to select for binding / affinity from a library of Composite Human Antibodies that are potential lead candidates. Composite Human Antibodies TM in clinical development Company Candidate Phase Gilead Simtuzumab (GS-6624) II* Opsona OPN-305 II Vascular Pharmaceuticals VPI-2690B Gilead GS-5745 I* NKT Therapeutics NKTT120 I Adheron Therapeutics SDP 051 * clinical trials in multiple indications correct as of 16 October 2014 II I 6

7 We have chosen to collaborate with Antitope because of their outstanding technology, long track record of successful humanisation projects, clear intellectual property position and peerless scientific knowledge of the antibody engineering space Maria Halasz Cellmid CEO Composite Proteins TM Antitope s Composite Protein technology is used to generate therapeutic proteins devoid of human T cell epitopes to minimise potential immunogenicity in patients without compromising protein activity. MHC class II binding eliminated T cell epitopes are removed from the protein by engineering mutations using our in silico tools in order to reduce or eliminate binding to human MHC class II molecules. Simultaneously, structure and homology analysis guides the targeting and substitution of key amino acids in order to retain the desired protein activity. % Donors responding WT Protein Advantages of Composite Proteins TM Composite Protein Confirmation of T cell epitope avoidance by Antitope s EpiScreen technology Excellent retention of functionality and specificity compared to the starting protein Proprietary technology, avoids other deimmunisation patents. Stable cell line and sample of lead Composite Protein TM provided as standard Day 6 Day 7 Day 8 Day 9 Applications Non-human proteins Composite Proteins can be produced using one or more human or non-human proteins as a reference. Immunogenicity Assessment Antibody & Protein Engineering Cell Line Development Antibody Drug Conjugates 7 New properties Novel Composite Proteins can be generated from protein libraries and screened for new properties. Different formats Composite Proteins can be generated in different formats including whole protein, protein fragments and fusion proteins. For more information [email protected] Optimisation of Pharmacokinetics

8 Cell Line Development Antitope offers a variety of cell line development packages to suite customers development needs, from microgram producing transient cell lines through to high expressing working cell banks ready to be passed to your GMP manufacturing partner. Cell lines are developed using Composite CHO (Chinese Hamster Ovary), NS0 (mouse myeloma) or SP2/0 cell lines, but other cell lines suitable for commercial manufacture can also be developed. Stable cell lines are grown in suspension and produce high yields of antibodies or proteins in chemically defined, serum-free medium. Transfection of the cells can be performed using our proprietary pant vector system. Our manufacturing cell lines have been successfully transferred to CMOs and manufacturers worldwide to produce cgmp material. Advantages of Antitopes cell line development packages High expression levels of desired antibodies and proteins NS0 and Sp2/0 cell lines available for biosimilar production Management of transfer to CMO for smooth transition to GMP production No downstream milestones or royalty payments Applications Generation of manufacturing cell lines for production of biopharmaceuticals suitable for GMP manufacture pant maximises recombinant protein expression through the use of a proprietary UTR (untranslated terminal region) to enhance protein production, and a modified dhfr gene for the rapid amplification of vector copy numbers in the cells. Protein purification up to 100L Cell lines can be cultured and expressed proteins or antibodies purified for use in preclinical studies. The cell lines can be derived from mammalian or bacterial sources. Purified proteins are supplied with a certificate of analysis indicating quantity and quality of material. Biosimilar development Antitope has experience of developing cell lines expressing biosimilars using NS0, Sp2/0 and CHO cell lines. 8

9 After a careful and diligent search for a partner capable of both meeting our exacting quality standards and delivering highly productive and compliant cgmp production cell lines, we are very happy to be working with Antitope on this important project. Additional Services 9 DNA sequencing Antitope s sequencing service supports a wide range of throughput and research application needs for DNA sequencing. We also apply internal insight and experience in sequencing hybridomas where transcripts are low or aberrant transcripts make sequencing difficult. Major Contract Manufacturing Organisation C EO Affinity maturation Affinity maturation is applied to improve antibody affinity for an antigen by mutation and selection against the desired antigen. Using phage display we can apply both hotspot mutagenesis using randomised codons and oligonucleotide-directed mutagenesis. Either method produces large numbers of variants which can be screened against antigen to select antibodies with improved affinity. SPR analysis can be performed on the best antibody leads as defined by the selection assay to determine accurate binding affinities. Molecular interaction analysis, including antibody affinity and protein-protein interaction assessment SPR analysis generates high quality data on the interactions between proteins and other molecules, including small molecule drug candidates. During research, development and manufacture, these data give insights into molecular function and disease mechanisms, and play a key role in the critical decisions needed for efficient development and production of therapeutics. Bespoke assay development We pride ourselves on our ability to work with our clients to develop the appropriate assay for their project. Should your therapeutic product require a bespoke or non-standard in vitro assay for assessment of activity at any stage of the development process, our scientific team has extensive experience in a huge range of cell- and plate-based assays, including cytotoxicity (ADCC and CDC), cell viability, proliferation and apoptosis assays; ADP-ribosylation and transcription assays; complement- and Fc receptor binding; luciferase reporter assays; dendritic cell phenotyping; cytokine release; ELISpot, flow cytometry, confocal microscopy, and many more. We can transfer in your existing assay or develop an assay de novo. For more information [email protected] Immunogenicity Assessment Antibody & Protein Engineering Cell Line Development Antibody Drug Conjugates Optimisation of Pharmacokinetics

10 Antibody Drug Conjugates PolyTherics has developed its range of novel ThioBridge linkers to efficiently conjugate drugs to antibodies to create less heterogeneous ADCs with better stability. Our proprietary technology uses site-specific conjugation to naturally occurring interchain disulfides avoiding the need for antibody re-engineering. ThioBridge TM ThioBridge conjugation is site-specific, highly stable and does not disrupt the tertiary structure of the protein. When ThioBridge has been used as part of the linker between an antibody and a cytotoxic payload the resulting ADC has been shown to have reduced heterogeneity and better stability compared to equivalent maleimide ADCs. Advantages of ThioBridge ADCs TM ADCs No need for re-engineering of antibodies as drug is conjugated to native disulfides Structural integrity of the antibody is retained as the disulfides are re-bridged More homogeneous ADCs produced with a majority (> 80%) DAR 4 Low risk of competitive inhibition of ADC binding as minimal DAR 0 produced Avoids undesirable high DAR ADCs Avoids instability associated with ADCs generated with maleimide reagents Applications of ThioBridge ADCs TM A Applicable to any antibody or protein format with accessible disulfides Applicable to a range of payloads with varying mechanisms of action Can be used with cleavable and non-cleavable linkers 10

11 11 This technology represents an exceptional strategic fit for ACT in our efforts to bring forward targeted cancer treatments which have the potential to significantly improve cancer care for many patients. ThioBridge TM ADCs are more homogenous with majority DAR 4 Thiobridge TM conjugation targets interchain disulfides. Comparison of the DAR distribution of ADCETRIS with an equivalent ThioBridge TM ADC; DAR was determined by HIC. The ThioBridge TM ADC was more homogeneous. ThioBridge TM ADCs have better stability than equivalent maleimide ADCs The same drug was conjugated to trastuzumab using ThioBridge or maleimide chemistry and conjugates purified to DAR 4 and then incubated in IgG-depleted serum for 120 h at 37 O C; hydrophobic interaction chromatography (HIC) was used to determine changes in DAR distribution. ThioBridge TM maintained 100% DAR4 whereas drug was lost from the maleimide conjugate. ThioBridge TM ADCs have been produced with different mabs, linkers and payloads DAR distribution of ADCs produced by conjugation of either MMAE (cleavable) or MMAF (noncleavable) to 3 different antibodies. ADCs were analysed using HIC; DAR4 was the predominant species with minimal amounts of unconjugated antibody (DAR0) for all ADCs % of total ThioBridge TM ThioBridge Maleimide ADC ThioBridge Maleimide Maleimide ADC PolyTherics offers product specific licences for its ThioBridge TM technology and also conducts bioconjugation studies for its partners. For more information [email protected] % DAR4 Igor Sherman Alpha Cancer Technologies CEO Trastuzumab SeaGen ThioBridge TM ADC ADCETRIS mab 1 mab 2 mab 3 mab 4 mab 1 mab 2 mab 3 mab 4 Cleavable MMAE DAR ratio Non -cleavable MMAF Immunogenicity Assessment Antibody & Protein Engineering Cell Line Development Antibody Drug Conjugates Optimisation of Pharmacokinetics

12 Optimisation of Pharmacokinetics PolyTherics provides a range of site-specific conjugation technologies to optimise the pharmacokinetics and pharmacodynamics of therapeutic peptides and proteins, including antibody fragments and other protein scaffolds, by extending their half-life to reduce the frequency of dosing. This improves patient compliance and could reduce the cost of treatment. PolyTherics also provides a low viscosity polymer, PolyPEG, to enable the easier manufacture and/or injection of conjugated proteins which need to be administered at high concentration. TheraPEG TM - disulfide conjugation TheraPEG is a technology for attaching polymers, such as polyethylene glycol (PEG), to proteins through site-specific conjugation at disulfide bonds. TheraPEG has the flexibility to conjugate a range of PEG formats, including linear and branched PEG and PolyTherics comb-shaped low viscosity polymer, PolyPEG Advantages of TheraPEG TM No protein engineering required as conjugate to native disulfides Disulfide is re-bridged to maintain structural integrity of the protein HiPEG TM - conjugation at polyhistidine HiPEG is a conjugation technology for site-specific conjugation of polymers at histidine sequences expressed within or at the N or C terminal ends of a protein. Conjugation at the terminus of the protein can have the advantage of reducing the shielding effect of the PEG on the functional part of the protein thereby minimising the effect on its activity. A histidine tag added to the terminus of a protein to facilitate purification can be used as the conjugation site without negative impact on purification methis utilising the poly-histidine tag. Advantages of HiPEG Histidine tags can minimise interference with protein binding Use of histidine tags removes the need for protein engineering 12

13 13 TheraPEG is a proven technology for extending the half-life and enhancing the beneficial characteristics of proteins and therefore enables our partners to develop better biopharmaceuticals to address significant clinical needs. John Burt Abzena CEO CyPEG TM - cysteine conjugation CyPEG is a conjugation technology for site-specific conjugation of polymers at a thiol side-chain on a free cysteine. The thiol residue on a cysteine readily undergoes selective and efficient conjugation. Proteins or peptides that do not have a cysteine can be engineered to provide a specific site for conjugation using CyPEG Advantages of CyPEG TM Avoids instability reported for maleimide conjugates Conjugation process is efficient over a wide range of ph conditions PolyPEG TM - modular polymer PolyPEG is a novel low viscosity polymer comprising a polymethacrylate backbone with short PEG side chains that form a comb-like structure which is conjugated to therapeutic proteins to extend their in vivo half-life. PolyPEG is available in a range of molecular weights achieved by varying the length of the backbone and the size of the PEG side chains. This enables the pharmacokinetics and pharmacodynamics of the conjugated protein to be optimised. Advantages of PolyPEG TM Alternative to linear and branched polymers where viscosity is an issue for manufacture or administration Modular architecture and range of conjugation chemistries available for optimal use PolyTherics offers product specific licences for its pharmacokinetic optimisation technologies and also conducts bioconjugation studies for its partners. For more information [email protected] Immunogenicity Assessment Antibody & Protein Engineering Cell Line Development Antibody Drug Conjugates Optimisation of Pharmacokinetics

14 Publications EpiScreen TM itope TM & TCED TM Baker MP et al., Immunogenicity of protein therapeutics: The key causes, consequences and challenges. Self NonSelf. 2010; 1(4): Baker MP and Carr FJ., Pre-clinical considerations in the assessment of immunogenicity for protein therapeutics. Curr Drug Saf. 2010; 5(4): Carr FJ and Baker MP., In vitro screening for antibody immunogenicity. In M. Little (Ed), Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009; pp Chester KA, Baker MP and Mayer A., Overcoming the immunologic response to foreign enzymes in cancer therapy. Expert Rev Clin Immunol. 2005; 1(4): Jaber A and Baker M., Assessment of the immunogenicity of different interferon beta-1a formulations using ex vivo T-cell assays. J Pharm Biomed Anal. 2007; 43(4): Joubert MK et al., Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses. J Biol Chem 2012; 287(30): Perry LCA, Jones TD and Baker MP., New approaches to prediction of immune responses to therapeutic proteins during preclinical development. Drugs R D. 2008; 9(6): Bryson CJ, Jones TD and Baker MP., Prediction of immunogenicity of therapeutic proteins: Validity of computational tools. Biodrug. 2010; 241:1-8 Barker. Current Drug Safety. 2010; 5(4): Advanced publication Ahmadi et al., Small Amounts of Sub-Visible Aggregates Enhance the Immunogenic Potential of Monoclonal Antibody Therapeutics; Pharmaceutical Research Composite Proteins TM Baker MP et al., Identification and removal of immunogenicity from therapeutic proteins. Curr Opin Drug Discov Devel. 2007; 10(2): Jones TD et al., Identification and removal of a promiscuous CD4+ T cell epitope from the C1 domain of factor VIII. J Thromb Haemost. 2005; 3(5): ThioBridge TM George Badescu, Penny Bryant et al., Bridging Disulfides for Stable and Defined Antibody Drug Conjugates Bioconjugate Chem., 2014, 25 (6), pp Optimisation of Pharmacokinetics Shaunak S et al., Site-specific PEGylation of native disulfide bonds in therapeutic proteins; Nature Chemical Biology 2012; 2: Khalili H, Godwin A et al., Comparative binding of disulfidebridged PEG-Fabs. Bioconjug Chem Nov 21;23(11): Da Pieve C et al., Modification of thiol functionalized aptamers by conjugation of synthetic polymers. Bioconjugate Chemistry. 2010; 21(1): Balan S et al., Site-specific PEGylation of protein disulfide bonds using a three-carbon bridge. Bioconjugate Chemistry. 2007; 18(1): Cong Y et al., Site-specific PEGylation at histidine tags. Bioconjugate Chemistry. 2012; 23(2): Badescu et al., A new reagent for stable thiol-specific conjugation. Bioconjugate Chemistry. 2014; 25(3): Composite Human Antibodies TM Holgate RG and Baker MP., Circumventing immunogenicity in the development of therapeutic antibodies. IDrugs 2009; 12(4): Jones TD et al., Deimmunization of monoclonal antibodies. Methods Mol Biol. 2009; 525:

15 Working with us Abzena offers a range of services and technologies to help you develop better antibodies and proteins. We have over 10 years of experience working with R&D scientists in a flexible and collaborative manner to reach project objectives. Our research groups work with you to perform specific projects or develop bespoke studies in the fields of immunogenicity assessment, protein engineering, cell line development and ADC development. We work on a fee for service basis and offer licences to our technologies when they are applied to your specific products. Please call us or us at [email protected] to arrange to speak to our technical experts about your requirements. Abzena plc, Antitope Limited & PolyTherics Limited Babraham Research Campus Babraham Cambridge CB22 3AT United Kingdom Tel: +44 (0) Some images are copyright Shutterstock

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