1. Cloned-transgenic farm animals produce a bispecific antibody for T-cell mediated tumor cell killing ISSAG 22-26 August 2005 VITERBO Rosa Minoia

2. Purpose of our study To produce a stable and self renewable bio-reservoir of tumor targeting single chain bispecific antibody Vectors carrying CDR (complementary-determining region) sequences of the bispecific antibody supra - agonist for CD28 on T cell (Ab1) and directed to a melanoma- associated proteoglycan (Ab2) were prepared for gene transfer in farm animal. Strategy

3. CD28 Human CD28 is an important costimulatory molecule found on all CD4+ Tcells and on about half of the CD8+ Tcells. T cell activities attributed to CD28 include prevention of anergy, induction of cytokine gene transcription, stabilization of cytokine mRNA andactivation of CD8+ cytotoxic T lymphocytes.

4. Here we take advantage of B cells as the native immunoglobulin factory and introduce pre-rearranged heavy and light chain variable genes of a bispecific single chain antibody (bi-ScFv) with melanoma x CD28 specificity (rM28) into transgenic rabbit. To decipher the relevant promoter sequences and intronic transcription elements that are essential for high-level expression of bi-scFv rM28, linearized constructs of three different expression-cassettes were microinjected into fertilized rabbit oocytes.

5. STRUCTURE OF THE MICROINJECTED RECOMBINANT DNA CONSTRUCTS The 9.3  CD28 scFv fragment with 5’ sequences of signal peptide L1 and L2 interspersed by intron are joined by a linker derived from elbow- region of the human Ig  CH 1 domain to the 9.2.27  melanoma scFv fragment with 3’ N-terminal human Ig  CL and c myc tag. Construct I and II share the same core but different size of their 3’ intronic sequence (  1-switch). However, both gene constructs generate transgenic rabbits with low level of bi-scFv rM28 expression.

6. We proceeded to place the wt-  -intron into a more “physiological” genomic environment: 3’ of the Vh-region of scFv1 and 5’ of the CH1-linker to scFv2(construct III) Previous work showed that the switch region located within the wt-  -intron of a human heavy chain gene increases the level of immunoglobulin transgene expression in mice but has a nominal effect in stably transfected myeloma cell lines. Optimized gene format for expression of bi-scFv rM28 in rabbit blood we used the splice gene construct III to generate cloned-transgenic calves.

7. EXPRESSION OF rM28 PROTEIN IN THE SERUM OF TRANSGENIC RABBITS. Construct I Construct II Construct III Symbols represent the interval of mg/L of protein in founder animals determined by flow cytometry analysis

10. To quantify the amount of bi-scFv rM28 and to monitor the expression levels in the blood at different time points, specific ELISA measurements were made of sera taken from cloned and control calves during the firs nine months post-partum.

11. To purify bi-scFv rM28 from animal blood, 80ml of sera from calf #407 rM28 were passed over a protein L agarose column and protein was eluted at pH3 in 0,1M glycine. 55 kDa The advantage of using calf serum for protein L affinity purification of bi-scFv rM28 is that the light chains of bovine immunoglobulin, unlike rabbit immunoglobulin do not interact with protein L. SDS-PAGE coomassie stain and immunoblotting indicate that the 55kDa bi-scFv rM28 derived from clones is of approximately 80 percent purify.

12. The binding capacities of sera of calf #407, and the purified rM28 standard were similar on Jukart T cells and SkMel63 melanoma cells. Mean fluorescence intensity of the gated cells incubated with sample and stained with polyclonal PE-labeled goat anti-mouse antibody (Dianova) Sera of calf #407 Purfied bi-scFv rM28 of calf #407 Bi-scFv rM28 standard Control calf serum

13. To investigate if the calf derived bi-scFv rM28 retains its biological function after purification we added samples of either the purified protein or source sera to co-cultures of human peripheral blood mononuclear cells (PBMC). TUMOR CELL DESTRUCTION WAS COMPLETE AFTER 3 DAYS OF CO-COLTURE WITH PBMC

14. CONCLUSION This novel gene farming approch underscores the principle that: BLOOD CELLS CAN BE DIRECTED TO PRODUCE HIGH CONCENTRATIONS OF FULLY ACTIVE THERAPEUTIC MOLECULES WITHOUT COMPROMISING THE ANIMAL’S NORMAL HEALTH, REPRODUCTION AND DEVELOPMENT AND WITHOUT INPUTS FROM FERMENTATION FACILITIES AND COSTLY TECHNICAL STAFF Future applications of transgene expression in blood extend to producing large quantities of additional immunoglobulin fusion proteins for treatment of autoimmunity, cancer and infection or for enzymatic and diagnostic use.

15. Acknowledgment Brem G Grosse-Hovest L Mueller S Wolf E Zakhartchenko V Besnfelder U Mueller M Thank you for your attention