Revolutionizing Cancer Diagnosis: Advanced Blood-Based BioMarker

1. Delhi . Mumbai . Bangalore . Pune . Indore BLOOD-BASED EPIGENETIC BIO MARKER OF HUMAN CANCER Identifying Partners/Licensees

2. BACKGROUND: MAGNITUDE OF CANCER: GLOBAL • Global cancer burden – 32.6 mill. • Incidence/year – 14.1 mill. (7.4 mill. ♂ + 6.7 mill. ♀) cancer patients • Leading cause of death • 8.2 mill. (13% of all human deaths/1 death every 5 sec) per year • ~ 65% of cancer-related death in low and middle income countries • African Region (5.4%), Eastern Mediterranean Region (3.7%), South East Asia Region (12.8%), and Western Pacific Region (29.7%) • Lack of access to basic health care – Diagnosis as well as Treatment • Low level of awareness in population • Current status • 70 % of 65+ year-old have >2 chronic diseases, including cancer (BMC Geriatrics, 2011) • Current WHO projection (World Cancer Report) • 26.4 mill. patients/17 mill. Deaths (1 death every 2 sec) in 2030 • Current GLOBAL projection for 2050 • Population of 80+ year-old to triple (Nature, 2014)

3. BACKGROUND: MAGNITUDE OF CANCER: INDIA • Av. life expectancy: 64 (♂) – 67 (♀) years • 2.5 mill. cancer cases (27 national registries) •  1.75 mill. (70%) in advance stage(s) •  0.55 mill. deaths every year (based on 2010 figure published in Lancet 2012) • Current estimate: ~ 1 mill. new cases/year • Projection 2020: ~ 1.15 mill. new cases/year • Not a ‘notifiable’ disease in India • Tobacco-related cancer – major cancer in India • Cause of ≥ 50% deaths: 42% men + 18% women • Majority of death (71%) in age group 30-70 years • Major cancer sites in men: lip, pharynx, stomach and lungs • Major cancer sites in women: cervix, stomach and breast • Only 25 Regional Cancer Centers in India • One RCC serving  45 mill. people

4. VISION STATEMENT - 1 • In the existing modality of cancer therapy, no convenient bio-marker is available to monitor progress of therapy and regression • No mid-term dose/course correction possible • Typical therapeutic protocol • Start a treatment course with a therapeutic agent • Await patient response/’feel good nod’ • Continue till the end • Discontinue, if adverse • Try some other therapeutic agent • Average dose/course • No individualized treatment possible

5. VISION STATEMENT - 2 • “Cancer is one of the most curable chronic diseases of humans” – Dr. Vincent T. deVita [Former Director, NCI and Yale Cancer Center (USA)] • Condition: Timely detection •  40 % of cancers can be prevented • Cancers of lungs, cervix, head & neck, GIT, etc. • Combating cancer by early detection •  35% enhanced clinical efficacy with existing therapeutic practice(s) • Significant mitigation of human suffering by ‘cancer screening’ • Screened population  Declining trend of cancer • Cancer statistics of USA for 2013 shows this • Incidence of cervical cancer in India

6. VISION STATEMENT - 3 • “Prevention is absolutely critical and it’s been somewhat neglected” • Dr. Chris Wild, Director, International Agency for Research on Cancer (IARC): BBC News/4th Feb. 2014 • Need a ‘good’ cancer bio-marker

7. INTRODUCTION TO TECHNOLOGY • Research spanning over two and a half decades has identified and characterized a novel and ideal biomarker of human cancer, which meets all three characteristics formulated by the international Predictive Safety Testing Consortium (PSTC) comprising industries, non-profit organizations and regulators (including FDA and EMEA/EMA). • A non-radioactive, highly sensitive, reproducible assay to quantitatively estimate the bio-marker using a minimally invasive quick process that does not require hospitalization has been developed. • The design of a complete and easy-to-use prototype for this test has already been developed in a diagnostic kit format that is highly reliable and portable to remote locations without compromising efficacy. • Phase-I clinical study undertaken in patients with 18 different cancers (grouped in head & neck, breast and cervical cancer categories) has provided statistically significant verification of the ‘proof of concept’.

8. NEED FOR TECHNOLOGY • Inadequacy of current therapeutic interventions for individualized mid-course corrections of ongoing therapy (change of drug, dose, strategy, etc.) reflects a serious gap in increasing clinical efficacy based on real-time quantitative biochemical or pathological tests. • The novel diagnostic kit developed fills this conspicuous gap as it can reliably and reproducibly detect multiple types of cancer using a minimally invasive OPD procedure requiring no hospitalization within a short time of less than 60 min and provide quantitative measurements of treatment efficacy and cancer retardation. • An ideal bio-marker of cancer can potentially be utilized for predictive bio-monitoring of cancer therapy to achieve higher clinical efficacy as well as for screening of populations for early detection of cancer. • The clinical efficacy of cancer therapy may be enhanced by 35 to 40% by monitoring the therapeutic progression using this sensitive and predictive biochemical test, presented in the format of a diagnostic kit, as proposed here.

9. NEED FOR TECHNOLOGY • Feedback from a large number of clinicians suggested that the existing clinical efficacy of cancer therapy can be enhanced greatly by: • (a) Making mid-course quantitative assessment of therapeutic effectiveness a reality, thus empowering the physician to take a decision based on quantitative information for required change of the ongoing therapeutic modality/strategy opening a corridor for individualized therapeutic course; • (b) Using a quick, reliable and predictive test that is minimally invasive, does not require hospitalization and can be carried out in remote locations with high success rates; and • (c) Early start of therapy due to early detection of cancer by using a screening test that is not limited to a specific tissue or type of cancer.

10. LIMITATIONS IN EXISTING TECHNOLOGIES • To the best of our knowledge, there is no other diagnostic kit or procedure for assessment of therapeutic status of cancer today that can match the projected position of technology being offered in terms of its spectrum of detection, sensitivity of the assay/test and ease of application (non-invasiveness, non-radioactive, non-hazardous nature of the components, quick result, no hospitalization, etc.). • The other bio-markers of cancer in use today are typically specific to a particular cancer or tissue type, the assays being time consuming and usually requiring hospitalization. • The scope of accessibility to these tests is expensive and limited only to select segments of populations with close proximity to large cities.

11. KEY ADVANTAGES • The novel technology has the potential to become a convenient general biomarker of cancer enabling reliable screening of remote populations for early detection of cancer without portability issues – an area that is currently void of any option, thus, expanding the umbrella for marketing of this diagnostic kit • Armed with quantitative information derived from this quick but sensitive test, it would become possible for the clinical oncologist to make necessary mid-term correction in therapy for clinical gains and make early detection of cancer in much larger segments of population a reality.

12. THE BIO-MARKER MOLECULE • ADP-ribose polymer (ARP) or poly-ADP-ribose (PAR) • Epigenetic in nature! • Highly heterogeneous polymer • Poly(ribose-nucleotide) • Molecular arrangement • Branched or unbranched • Different size • Multiple architecture • Multiple location • Multiple medical applications • Biomarker of human cancer • Biomonitor of cancer therapeutics • Predictive diagnosis

13. METABOLIC PROCESS • Post-translation modification of proteins • Enzyme catalyzed reaction • Fully reversible • Preferred target – histone proteins • Heterogeneous, branched/unbranched polymer of ‘ADP-ribose’ units of variable lengths • Target amino acid residues: Arg & Lys • 2 –ve charges/monomer added on target proteins • DNA-protein charge interaction(s) altered • Growing physical bulk of the polymer • Conformational and functional status of chromosome strongly influenced

14. SUMMARY OF TECHNOLOGY WHEN TESTED ON MICE • ADP-ribose polymer: indicator of carcinogenesis • Initiation phase • Promotion phase • Progression phase • Carcinogenesis induction • DEN, DMN, Arecoline, Betel nut • Ascites • Target as well as non-target tissue/proteins • Liver, Spleen cells, BMC, other tissues • Total cellular proteins • Histone proteins, HMG proteins • Blood lymphocytes mirror the status

15. PROOF OF CONCEPT Phase 1 study: Human Cancers • Cancers under study n = 111 • Breast n = 22 • Cervix n = 24 • Head & Neck (14 different cancers/sites) n = 65 • Advance stage cancers • ADP-ribose polymer quantified in peripheral blood lymphocytes (PBL) • 2 ml blood collected • PBL isolated, homogenized, blotted and immunoprobed • Controls: no known history of cancer n = 68 • Mainly univ. students (volunteers) • Relatives • Males and females

16. PROOF OF CONCEPT Breast and Cervical Cancers: Total cellular ARP in cancer of breast compared to control [Number of individuals: Controls: All = 68, females = 27, breast cancer patients = 22]. Total cellular ARP in cancer of cervix compared to control [Number of individuals: Controls: All = 68 & females = 27, cervix cancer = 24].

17. PROOF OF CONCEPT Head and Neck Cancer: Total cellular ARP in cancers of head and neck compared to control [Number of individuals: Controls = 68, Ca Esophagus = 19, Alveolo = 6, Pyriform sinus = 4, Nasopharynx = 10, Larynx = 1, Tongue = 4, Tonsil = 5, Buccal mucosa = 3, Pharynx = 3, Lip = 3, Nasal cavity = 2, Oral cavity = 2, Epiglottis = 4, Vocal cord = 1].

18. PROOF OF CONCEPT All Cancers- Age and Gender Matched

19. PROOF OF CONCEPT 2nd level R&D – Direct ‘Immuno-probe assay’ Man Blood from finger tip Mouse spleen cell Lysis of blood Heterogeneous ADP-ribose polymer (ARP) Adsorption of lysed blood on NCM Purification & characterization Wash Raise Polyclonal anti-APR in rabbit ELISA based DIRECTimmunoprobing using polyclonal anti-PAR~HRP/India ink stain for total protein Purification of antiserum (PAb) Polyclonal Ab~HRP Ag (PAR) on target Color development Lyophilize PAb PAb conjugated with HRP/ALP Quantification using a digital densitometer PAR of total cellular proteins in blood PAb~HRP/ALP solution

20. THE KIT IN MAKING ADP-Ribose polymer mediated Rapid and Easy test for human Cancer (‘ARREST – C’)

21. THE KIT IN MAKING 0.75 ml 0.5 ml 0.3 ml 0.5 ml 0.3 ml 0.3 ml Cut-out Volumes 0.5 ml Lysis Buffer Blood Sample Wash buffer Ab-HRP Wash buffer Colour Developer Water Lysis Buffer

22. CONCLUSIONS • ADP-ribose polymer: a potential biomarker of human cancer • Useful in prognosis & diagnosis • Biomonitor of therapeutic progression • Potentially usable for screening population(s) • Multi-center human study is required • Phase II clinical study to be initiated • The novel immunoprobe assay of PAR to be packaged in a kit

23. PATENT/IP STATUS • Application Number: 1791/KOL/2008 • Application Status: Patent Pending • Priority Date: 23/10./2008

24. CONTACT DETAILS Delhi . Mumbai . Bangalore . Pune . Indore Contact Details Noida (NCR) Office E-13, UPSIDC Site-IV, Behind Grand Venice, Greater Noida, 201308 Contact Person: Tarun Khurana Contact No.: +91-120-2342010-11/9810617992 E-Mail: iiprd@iiprd.com,info@khuranaandkhurana.com Website: www.iiprd.com | www.khuranaandkhurana.com