The document discusses the development and benefits of cryopreserved human peripheral blood mononuclear cell (PBMC) libraries with defined HLA types and antigen reactivities. Key points include: 1) Protocols were established for functionally loss-free freezing and thawing of PBMCs. 2) A reference PBMC library was created with over 70 donors characterized for HLA type and immune responses. 3) Serum-free media was developed and implemented for PBMC processing and testing. 4) The libraries provide standardized access to characterized PBMCs for immunological research and assays.

1. Cryopreserved human PBMC libraries with defined HLA-type and antigen / peptide-reactivity for accelerating and standardizing human immunological research. (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

2. Step 1 towards standardization: Functionally loss-free freezing of PBMC In 2003 CTL scientists introduced protocols for functionally loss-free freezing of PBMC: Kreher et al. : CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays. J. Immunol. Methods, 278:79-93, 2003 (IFN- γ , IL-2, IL-4 and IL-5 were measured) (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

3. 1a. The role of temperature: Blood should be shipped, stored, and processed all times at room temperature, not chilled. For freezing, the cells and the DMSO-containing cryomedium must be at room temperature (not ice-cold) when mixed. When thawing, bring cells rapidly to 37 o C. (c) 2009 Cellular Technology Limited - CONFIDENTIAL - Live Apoptotic Dead

4. 1b. Following this protocol, we found that high and low frequency, high and low avidity T cells are preserved, loss free. No “resting” needed after thawing. (c) 2009 Cellular Technology Limited - CONFIDENTIAL - NIAID Contr. HHSN266200400098I, ELISPOT Qualification, Vaccinia Ex vivo/ Ex cryo

5. Since 2005, CTL offers a reference PBMC library, presently consisting of over 70 PBMC donors to chose from, each High resolution HLA-typed (Class I and Class II) Immune-characterized for 20 antigens, recognized by CD8 and CD4 cells IFN- γ , IL-2, IL-4, IL-5, IL-17, Granzyme B High and low frequency T cell responses High and low avidity T cell responses Essentially unlimited availability of identical cell material: between 500 and 1,000 aliquots of 10 million PBMC/vial from each donor available from each draw. Reference PBMC are essential for assay comparisons, qualification/ validation, and harmonization across institutions (c) 2009 Cellular Technology Limited - CONFIDENTIAL - Step 2 towards standardization: Reference PBMC

6. Example: response profile of donor 19 (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

7. In 2005, CTL introduced serum free media for all steps of PBMC processing and testing Freezing (CTL-Cryo™) Thawing (CTL-Deaggregate ™) Washing (CTL-Wash ™) Testing (CTL-Test ™) Since, we and our customers have seen no serum that performs significantly better, but many sera that perform (much) worse. (c) 2009 Cellular Technology Limited - CONFIDENTIAL - Step 3 towards standardization: Serum Free Media

8. Wouldn't if be fantastic if you... Did not need to chase your colleagues with a needle anymore to obtain some peripheral blood mononuclear cells (PBMC) for your experiments? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

9. Wouldn't if be fantastic if you... Did not need to bother with IRBs just to obtain some blood from healthy volunteers because we have taken care of this for you? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

10. Wouldn't if be fantastic if you... Can do experiments that you have never done before because you can obtain as many PBMC as you need (up to 10 billion cells from the same donor at the same time of bleed)? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

11. Wouldn't if be fantastic if you... Can immediately repeat experiments with PBMC of the same donor and of the same bleed, if the reviewer asks you to provide that one additional control? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

12. Wouldn't if be fantastic if you... Can share the same PBMC with collaborators anywhere on the planet? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

13. Wouldn't if be fantastic if you... Can prepare PBMC for an experiment more simply and more quickly than isolating such cells from blood by yourself? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

14. Wouldn't if be fantastic if you... Have immediate access to a library of PBMC that are pre-characterized, with established HLA-types and antigen reactivities? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

15. Wouldn't if be fantastic if you... Can design your experiments by selecting test subjects from a panel of characterized human donors? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

16. Wouldn't if be fantastic if you... Can use such pre-characterized cells as positive and negative controls for any immune monitoring effort, and as reference samples to control the consistent performance of your assays? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

17. Wouldn't if be fantastic if you... Have access to a large number of human donors that you can screen for an immune reactivity and/or genetic marker of your own individual interest? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

18. Wouldn't if be fantastic if you... Do not have to worry about the human material you work with is HIV , Hepatitis B, etc infected? (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

19. CTL has made this dream come true by offering immune characterized cryopreserved PBMC libraries! (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

20. (c) 2009 Cellular Technology Limited - CONFIDENTIAL - Identifying reference PBMC samples Utilization of ePBMC for ELISPOT assay development, qualification and validation Single color human, murine and macaque IFN- γ assay, Vaccinia/MVA- specific NIAID ContrNo. HHSN266200400098I, highlights

21. (c) 2009 Cellular Technology Limited - CONFIDENTIAL - a. Optimal antigen dose

22. b. Accuracy (c) 2009 Cellular Technology Limited - CONFIDENTIAL -

23. c. Linearity (and range) (c) 2009 Cellular Technology Limited - CONFIDENTIAL - r=0.998

24. (c) 2009 Cellular Technology Limited - CONFIDENTIAL - d. Precision – Inter assay repeatability

25. (c) 2009 Cellular Technology Limited - CONFIDENTIAL - e. Precision Intermediate precision (using low responder)

26. (c) 2009 Cellular Technology Limited - CONFIDENTIAL - Kinetics of cytokine production after CMV stimulation

27. (c) 2009 Cellular Technology Limited - CONFIDENTIAL - Linearity of CMV induced cytokine spots TNTC Number of PBMC per Well SFU per Well