Anti-P. falciparum AMA1 Therapeutic Antibody (14M-15)

Figure 1 V(NAR) Library Design

ELISA testing of affinity-purified VNAR proteins (14I-1, 14M-15, 12A-9) for cross-strain AMA1 binding. Data are represented as the mean ± standard error of triplicate experiments.

Henderson, K. A., Streltsov, V. A., Coley, A. M., Dolezal, O., Hudson, P. J., Batchelor, A. H., ... & Foley, M. (2007). Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure, 15(11), 1452-1466.

Figure 2 Affinity maturation of V(NAR) 12Y-2 by error-prone PCR.

BIAcore sensorgrams comparing the binding of identical concentrations of the parental and mutant proteins to immobilized AMA1 at a constant flow rate of 5 ul/min with an injection volume of 35 ul.

Nuttall, S. D., Humberstone, K. S., Krishnan, U. V., Carmichael, J. A., Doughty, L., Hattarki, M., ... & Irving, R. A. (2004). Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1. PROTEINS: Structure, Function, and Bioinformatics, 55(1), 187-197.

Figure 3 The 12Y-2 triple-mutant shows parental affinity for AMA1.

BIAcore sensorgrams comparing the binding of identical concentrations of the parental (12Y-2), single mutant (14M-15), and triple mutant proteins to immobilized AMA1.

Nuttall, S. D., Humberstone, K. S., Krishnan, U. V., Carmichael, J. A., Doughty, L., Hattarki, M., ... & Irving, R. A. (2004). Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1. PROTEINS: Structure, Function, and Bioinformatics, 55(1), 187-197.

Figure 4 Tandem V(NAR)s in a single chain format

BIAcore sensorgrams comparing binding of proteins 12Y-2, 14M-15, and 15Q-1 to immobilised AMA1 protein.

Simmons, D. P., Abregu, F. A., Krishnan, U. V., Proll, D. F., Streltsov, V. A., Doughty, L., ... & Nuttall, S. D. (2006). Dimerisation strategies for shark IgNAR single domain antibody fragments. Journal of immunological methods, 315(1-2), 171-184.

Figure 5 ELISA analysis of 12 14M-15 variants (12Y-2 with Pro90Leu) for binding to AMA1 and a control negative antigen.

ELISA analysis of 12 14M-15 variants (12Y-2 with Pro90Leu) for binding to AMA1 and a control negative antigen. Comparative expression levels are also shown. The CDR1 analogous region (loop region 4) has been shuffled in the variants.

Figure 6 ELISA analysis of 40 12Y-2 variants binding to AMA-1 and a control negative antigen.

ELISA analysis of 40 12Y-2 variants binding to AMA-1 and a control negative antigen.

Figure 7 Biosensor traces of purified 12Y-2 variant proteins 14M-15 and 22A-2. 22A-2 shows 2-fold enhanced affinity over 14M-15 (20-fold better affinity than 12Y-2).

Biosensor traces of purified 12Y-2 variant proteins 14M-15 and 22A-2. 22A-2 shows 2-fold enhanced affinity over 14M-15 (20-fold better affinity than 12Y-2).

Figure 8 V(NAR) 14M-15 targets a variable region of P. falciparum AMA1.

ELISA comparison of V(NAR) protein 14M-15 binding to AMA1 derived from P. falciparum strains W2MEF, HB3, D10, and 3D7. Binding to reduced and alkylated 3D7 (R&A) is also shown.

Nuttall, S. D., Humberstone, K. S., Krishnan, U. V., Carmichael, J. A., Doughty, L., Hattarki, M., ... & Irving, R. A. (2004). Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1. PROTEINS: Structure, Function, and Bioinformatics, 55(1), 187-197.

Figure 9 Parasite Invasion Inhibition

Recombinant V(NAR) domains inhibit invasion of erythrocytes by P. falciparum 3D7 parasites. Data are represented as the mean ± standard error of quadruplicate experiments.

Henderson, K. A., Streltsov, V. A., Coley, A. M., Dolezal, O., Hudson, P. J., Batchelor, A. H., ... & Foley, M. (2007). Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure, 15(11), 1452-1466.

Figure 10 Parasite Invasion Inhibition

Recombinant V(NAR) domains inhibit invasion of erythrocytes by P. falciparum 3D7 parasites. Data are represented as the mean ± standard error of quadruplicate experiments,except for P. falciparum W2mef parasites.

Henderson, K. A., Streltsov, V. A., Coley, A. M., Dolezal, O., Hudson, P. J., Batchelor, A. H., ... & Foley, M. (2007). Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure, 15(11), 1452-1466.

Figure 11 V(NAR) Library Design

ELISA testing of affinity-purified V(NAR) proteins (14I-1, 14M-15, 12A-9) for cross-strain AMA1 binding.

Henderson, K. A., Streltsov, V. A., Coley, A. M., Dolezal, O., Hudson, P. J., Batchelor, A. H., ... & Foley, M. (2007). Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure, 15(11), 1452-1466.