EA043756B1 - ANTI-PFRH5 ANTIBODIES AND THEIR ANTIGEN-BINDING FRAGMENTS - Google Patents

Description

Field of technology

This invention relates in part to antibodies and antigen-binding fragments thereof that specifically bind to PfRH5, as well as methods of using them for the treatment or prevention of infections caused by Plasmodium falciparum.

State of the art

Invasion of host erythrocytes represents an important step in the life cycle of Plasmodium falciparum and malaria pathology. Many antimalarial drugs target asexual stages in the bloodstream, but their effectiveness is threatened by the emergence of drug-resistant strains (Arrow et al., Saving Lives, Buying Time: Economics of Malaria Drugs in an Age of Resistance. National Academies Press (US) 254-266 (2004) PMID: 25009879; and Wright et al., Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies, Nature: 515:427-430 (2014) PMID: 25132548. In addition, antimalarial drugs have different pharmacokinetic properties. Some antimalarial drugs, such as artemisinin and quinine, are rapidly cleared from the body within one parasite's life cycle. On the other hand, hydrophobic and lipophilic antimalarials are eliminated slowly, but they exhibit different rates of absorption depending on the amount of fat consumed (Arrow et al.).

Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5) is a member of a superfamily of erythrocyte ligands called reticulocyte binding-like proteins (RBLs). PfRH5 binds erythrocytes, is likely required for parasite growth at the bloodstream stage, and is involved in the species tropism of erythrocyte invasion. Evidence suggests that the receptor for PfRH5 on erythrocytes is the Ok blood group antigen, basigin (BSG; CD147).

Brief description of the invention

The present invention provides an antigen binding protein (eg, an antibody or an antigen binding fragment thereof) that (i) specifically binds to the same epitope on Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) as; or (ii) competes for binding to a PfRH5 polypeptide with: an antibody or an antigen-binding fragment thereof that contains (a) an immunoglobulin heavy chain that contains CDR-H1, CDR-H2 and CDR-H3 of an immunoglobulin heavy chain that contains the amino acid sequence of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354; and/or (b) an immunoglobulin light chain that contains CDR-L1, CDR-L2 and CDR-L3 of an immunoglobulin light chain that contains the amino acid sequence of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122 , 138, 154, 170, 186, 202, 218, or 306. For example, in an embodiment of the present invention, the anti-PfRH5 antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises (i) an immunoglobulin heavy chain that contains CDR-H1, CDR-H2 and CDR-H3 of the immunoglobulin heavy chain, which contains the amino acid sequence SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234 , 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354; and/or (ii) an immunoglobulin light chain that contains CDR-L1, CDR-L2 and CDR-L3 of an immunoglobulin light chain that contains the amino acid sequence of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122 , 138, 154, 170, 186, 202, 218, or 306. For example, in an embodiment of the present invention, an antigen binding protein (e.g., an antibody or an antigen binding fragment thereof) comprises: (a) an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242 , 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354; and/or (b) an immunoglobulin light chain variable region comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122 , 138, 154, 170, 186, 202, 218, or 306. In an embodiment of the present invention, the antigen binding protein (e.g., an antibody or an antigen binding fragment thereof) comprises (a) an immunoglobulin heavy chain containing CDR-H1, CDR-H2, and CDR- H3 immunoglobulin heavy chain containing the amino acid sequence SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354, and having at least 90% amino acid sequence identity with the amino acid sequence set forth in NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242, 250,

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258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354; and/or (b) an immunoglobulin light chain comprising CDR-L1, CDR-L2 and CDR-L3 of an immunoglobulin light chain comprising the amino acid sequence SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138 , 154, 170, 186, 202, 218 or 306, and having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138 , 154, 170, 186, 202, 218, or 306. This invention also includes an antigen binding protein (e.g., an antibody or an antigen binding fragment thereof) comprising: an immunoglobulin heavy chain that contains a CDR-H1 containing the amino acid sequence: GYSFTSYW (SEQ ID NO : 4); CDR-H2 containing the amino acid sequence: IYPGDSDT (SEQ ID NO: 6); and CDR-H3 containing the amino acid sequence: ARQDITGTTGFDY (SEQ ID NO: 8); or CDR-H1 containing the amino acid sequence: GFTFSSYA (SEQ ID NO: 20); CDR-H2 containing the amino acid sequence: ISYDGSNK (SEQ ID NO: 22); and CDR-H3 containing the amino acid sequence: AKERLFGVVSYYGMDV (SEQ ID NO: 24); or CDR-H1 containing the amino acid sequence: GGSISSSSYY (SEQ ID NO: 36); CDR-H2 containing the amino acid sequence: IYYSGST (SEQ ID NO: 38); and CDR-H3 containing the amino acid sequence: ARQDREALFDY (SEQ ID NO: 40); or CDR-H1 containing the amino acid sequence: GFRFDDYA (SEQ ID NO: 52); CDR-H2 containing the amino acid sequence: I NWNSGGK (SEQ ID NO: 54); and CDR-H3 containing the amino acid sequence: A KDRGIAARLLSRDAFDM (SEQ ID NO: 56); or CDR-H1 containing the amino acid sequence: SFTFSSYG (SEQ ID NO: 68); CDR-H2 containing the amino acid sequence: ISYDGSNK (SEQ ID NO: 70); and CDR-H3 containing the amino acid sequence: AREVRRYYYYGMD

V (SEQ ID NO: 72); or CDR-H1 containing the amino acid sequence: GF T F D D Y A (SEQ ID NO: 84); CDR-H2 containing the amino acid sequence: ISWNSGDI (SEQ ID NO: 86); and CDR-H3 containing the amino acid sequence: AKDTLSGTGTTWYYFDY (SEQ ID NO: 88); or CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 100); CDR-H2 containing the amino acid sequence: ISYDGSNK (SEQ ID NO: 102); and CDR-H3 containing the amino acid sequence: AQDGSSAIYYFYGMDV (SEQ ID NO: 104); or CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 116); CDR-H2 containing the amino acid sequence: IWYDGSNK (SEQ ID NO: 118); and CDR-H3 containing the amino acid sequence: ARGEHYYGSGPFDP (SEQ ID NO: 120); or CDRH1 containing the amino acid sequence: GGSISSFG

YY (SEQ ID NO: 132); CDR-H2 containing the amino acid sequence: IYYSGSI (SEQ ID NO: 134); and CDR-H3 containing the amino acid sequence: ARERDYGDYFDY (SEQ ID NO: 136); or CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 148); CDR-H2 containing the amino acid sequence: IWYDGSNK (SEQ ID NO: 150); and CDR-H3 containing the amino acid sequence: ARDQDYYGSGSSYGMDV (SEQ ID NO: 152); or CDR-H1 containing the amino acid sequence: GFT

FSTYG (SEQ ID NO: 164); CDR-H2 containing the amino acid sequence: IWYDGTNK (SEQ ID NO: 166); and CDR-H3 containing the amino acid sequence: ARDPSGGDHYYYYGMDV (SEQ ID NO: 168); or CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 180); CDR-H2 containing the amino acid sequence: ISFDERNK (SEQ ID NO: 182); and CDR-H3 containing the amino acid sequence: ASEVGYSFGH DAFDI (SEQ ID NO: 184); or CDR-H1 containing the amino acid sequence: GFTFNNYA (SEQ ID NO: 196); CDR-H2 containing the amino acid sequence: ISGSGDST (SEQ ID NO: 198); and CDR-H3 containing the amino acid sequence: AKDQGLYYYGSGSFDY (SEQ ID NO: 200); or CDR-H1 containing the amino acid sequence: GFAFSDSA (SEQ ID NO: 212); CDR-H2 containing the amino acid sequence: IRNKANRFAT (SEQ ID NO: 214); and CDR-H3 containing the amino acid sequence: ARHGHDTLTEGYGMDV (SEQ ID NO: 216); or CDR-H1 containing the amino acid sequence: GGTFSSYT (SEQ ID NO: 228); CDR-H2 containing the amino acid sequence: IIPLYGTA (SEQ ID NO: 230); and CDR-H3 containing the amino acid sequence: ASTLELRAFDAFDI (SEQ ID NO: 232); or CDR-H1 containing the amino acid sequence: GGSISSGGYY (SEQ ID NO: 236); CDR-H2 containing the amino acid sequence: IYYSGST (SEQ ID NO: 238); and CDR-H3 containing the amino acid sequence: ARAPPYNWFDY (SEQ ID NO: 240); or CDR-H1 containing the amino acid sequence: GFTFSDYY (SEQ ID NO: 244); CDR-H2 containing the amino acid sequence: ISNSGNTQ (SEQ ID NO: 246); and CDR-H3 containing the amino acid sequence: TREGLEYSSSEPFDY (SEQ ID NO: 248); or CDR-H1 containing the amino acid sequence: GYTFTAYY (SEQ ID NO: 252); CDR-H2 containing the amino acid sequence: INPNNGDT (SEQ ID NO: 254); and CDR-H3 containing the amino acid sequence: ARDDLAAAGIGWFDS (SEQ ID NO: 256); or CDR-H1 containing the amino acid sequence: GFTFDDYA (SEQ ID NO: 260); CDR-H2 containing the amino acid sequence: ISWNSESI (SEQ ID NO: 262); and CDR-H3 containing the amino acid sequence:

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AKAPYSGTYFEYFRH (SEQ ID NO: 264); or CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 268); CDR-H2 containing the amino acid sequence: ISYDGSNK (SEQ ID NO: 270); and CDR-H3 containing the amino acid sequence: AKDDWNYDAFDI (SEQ ID NO: 272); or CDR-H1 containing the amino acid sequence: GGSISSSGYY (SEQ ID NO: 276); CDR-H2 containing the amino acid sequence: IYYSGST (SEQ ID NO: 278); and CDR-H3 containing the amino acid sequence: ARVDYGSGSSFDY (SEQ ID NO: 280); or CDR-H1 containing the amino acid sequence: GYTFTSYG (SEQ ID NO: 284); CDR-H2 containing the amino acid sequence: ISGFNGRT (SEQ ID NO: 286); and CDR-H3 containing the amino acid sequence: ARDGLEKLGDY (SEQ ID NO: 288); or CDR-H1 containing the amino acid sequence: GFTFSNSG (SEQ ID NO: 292); CDR-H2 containing the amino acid sequence: IWHDGSYK (SEQ ID NO: 294); and CDR-H3 containing the amino acid sequence: ARDDYYASGTSVDV (SEQ ID NO: 296); or CDR-H1 containing the amino acid sequence: GYTFTGYY (SEQ ID NO: 300); CDR-H2 containing the amino acid sequence: INPNSGGT (SEQ ID NO: 302); and CDR-H3 containing the amino acid sequence: AREEVDDFWSGYLDY (SEQ ID NO: 304); or CDR-H1 containing the amino acid sequence: GFAVNGDY (SEQ ID NO: 316); CDR-H2 containing the amino acid sequence: IYSSGNT (SEQ ID NO: 318); and CDR-H3 containing the amino acid sequence: ARDFPPMSGADY (SEQ ID NO: 320); or CDR-H1 containing the amino acid sequence: GYTLTELS (SEQ ID NO: 324); CDR-H2 containing the amino acid sequence: FDPEHGKI (SEQ ID NO: 326); and CDR-H3 containing the amino acid sequence: A TFYNWNSYYFGMDV (SEQ ID NO: 328); or CDR-H1 containing the amino acid sequence: GFTFSSYA (SEQ ID NO: 332); CDR-H2 containing the amino acid sequence: VSGSADIT (SEQ ID NO: 334); and CDR-H3 containing the amino acid sequence: AKDKVYNWNYGIYYGMDV (SEQ ID NO: 336); or CDR-H1 containing the amino acid sequence: GGSISSSSYY (SEQ ID NO: 340); CDR-H2 containing the amino acid sequence: IYYSGST (SEQ ID NO: 342); and CDR-H3 containing the amino acid sequence: ARQGRWERENFDY (SEQ ID NO: 344); or CDR-H1 containing the amino acid sequence: DESFSDYY (SEQ ID NO: 348); CDR-H2 containing the amino acid sequence: ITHSGST (SEQ ID NO: 350); and CDR-H3 containing the amino acid sequence: ARGGDYGGLLDY (SEQ ID NO: 352); and/or an immunoglobulin light chain variable region, which contains CDR-L1 containing the amino acid sequence: QSIRNY (SEQ ID NO: 12); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 14); and CDR-L3 containing the amino acid sequence: QQSYSTPFT (SEQ ID NO: 16); or CDR-L1 containing the amino acid sequence: QDINRD (SEQ ID NO: 28); CDR-L2 containing the amino acid sequence: DAS (SEQ ID NO: 30); and CDR-L3 containing the amino acid sequence: QQYKNLPYT (SEQ ID NO: 32); or CDR-L1 containing the amino acid sequence: QRIGSS (SEQ ID NO: 44); CDR-L2 containing the amino acid sequence: YAS (SEQ ID NO: 46); and CDR-L3 containing the amino acid sequence: HQSSTLPT (SEQ ID NO: 48); or CDR-L1 containing the amino acid sequence: QDVSSY (SEQ ID NO: 60); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 62); and CDR-L3 containing the amino acid sequence: QHLNTYPYT (SEQ ID NO: 64); or CDR-L1 containing the amino acid sequence: QDISNY (SEQ ID NO: 76); CDR-L2 containing the amino acid sequence: DAS (SEQ ID NO: 78); and CDR-L3 containing the amino acid sequence: QQYNNLPLT (SEQ ID NO: 80); or CDR-L1 containing the amino acid sequence: QGISSY (SEQ ID NO: 92); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 94); and CDR-L3 containing the amino acid sequence: QQVNSYPLT (SEQ ID NO: 96); or CDRL1 containing the amino acid sequence: QDINNY (SEQ ID NO: 108); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 110); and CDR-L3 containing the amino acid sequence: LQYNSYHPT (SEQ ID NO: 112); or CDR-L1 containing the amino acid sequence: QSISNY (SEQ ID NO: 124); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 126); and CDR-L3 containing the amino acid sequence: QQSYSSPLT (SEQ ID NO: 128); or CDR-L1 containing the amino acid sequence: QSVSSN (SEQ ID NO: 140); CDR-L2 containing the amino acid sequence: GAS (SEQ ID NO: 142); and CDR-L3 containing the amino acid sequence: QQYNNWPLT (SEQ ID NO: 144); or CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 156); CDRL2 containing the amino acid sequence: AAS (SEQ ID NO: 158); and CDR-L3 containing the amino acid sequence: QQSYSTPLT (SEQ ID NO: 160); or CDR-L1 containing the amino acid sequence: QDISNY (SEQ ID NO: 172); CDR-L2 containing the amino acid sequence: DAS (SEQ ID NO: 174); and CDR-L3 containing the amino acid sequence: QQYDNIPIT (SEQ ID NO: 176); or CDR-L1 containing the amino acid sequence: QDISNY (SEQ ID NO: 188); CDR-L2 containing the amino acid sequence: DAS (SEQ ID NO: 190); and CDR-L3 containing the amino acid sequence: QQYDNFPLT (SEQ ID NO:

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192); or CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 204); CDRL2 containing the amino acid sequence: AAS (SEQ ID NO: 206); and CDR-L3 containing the amino acid sequence: QQSYSTPFT (SEQ ID NO: 208); or CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); or CDR-L1 containing the amino acid sequence: QSVSSSY (SEQ ID NO: 308); CDR-L2 containing the amino acid sequence: GAS (SEQ ID NO: 310); and CDR-L3 containing the amino acid sequence: QQYGSSPWT (SEQ ID NO: 312). In one embodiment of the present invention, an antigen binding protein (eg, an antibody or an antigen binding fragment thereof) comprises (1) an immunoglobulin heavy chain variable region that contains a CDR-H1 containing the amino acid sequence: GYSFTSYW (SEQ ID NO: 4); CDR-H2 containing the amino acid sequence: IYPGDSDT (SEQ ID NO: 6); and CDR-H3 containing the amino acid sequence: ARQDITGTTGFDY (SEQ ID NO: 8); and an immunoglobulin light chain variable region, which contains CDR-L1 containing the amino acid sequence: QSIRNY (SEQ ID NO: 12); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 14); and CDR-L3 containing the amino acid sequence: QQSYSTPFT (SEQ ID NO: 16); (2) an immunoglobulin heavy chain variable region, which contains a CDR-H1 containing the amino acid sequence: GFTFSSYA (SEQ ID NO: 20); CDR-H2 containing the amino acid sequence: ISYDGSNK (SEQ ID NO: 22); CDR-H3 containing the amino acid sequence: AKERLFGVVSYYGMDV (SEQ ID NO: 24); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QDINRD (SEQ ID NO: 28); CDR-L2 containing the amino acid sequence: DAS (SEQ ID NO: 30); and CDR-L3 containing the amino acid sequence: QQYKNLPYT (SEQ ID NO: 32); (3) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GGSISSSSYY (SEQ ID NO: 36); CDR-H2 containing the amino acid sequence: IYYSGST (SEQ ID NO: 38); and CDR-H3 containing the amino acid sequence: ARQDREALFDY (SEQ ID NO: 40); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QRIGSS (SEQ ID NO: 44); CDR-L2 containing the amino acid sequence: YAS (SEQ ID NO: 46); and CDR-L3 containing the amino acid sequence: HQSSTLPT (SEQ ID NO: 48); (4) an immunoglobulin heavy chain variable region, which contains a CDR-H1 containing the amino acid sequence: GFRFDDYA (SEQ ID NO: 52); CDR-H2 containing the amino acid sequence: INWNSGGK (SEQ ID NO: 54); and CDR-H3 containing the amino acid sequence: AKDRGIAARLLSRDAFDM (SEQ ID NO: 56); and an immunoglobulin light chain variable region, which contains CDR-L1 containing the amino acid sequence: QDVSSY (SEQ ID NO: 60); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 62); and CDR-L3 containing the amino acid sequence: QHLNTYPYT (SEQ ID NO: 64); (5) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: SFTFSSYG (SEQ ID NO: 68); CDR-H2 containing the amino acid sequence: ISYDGSNK (SEQ ID NO: 70); and CDR-H3 containing the amino acid sequence: AREVRRYYYYGMDV (SEQ ID NO: 72); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QDISNY (SEQ ID NO: 76); CDR-L2 containing the amino acid sequence: DAS (SEQ ID NO: 78); and CDR-L3 containing the amino acid sequence: QQYNNLPLT (SEQ ID NO: 80); (6) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFTFDDYA (SEQ ID NO: 84); CDR-H2 containing the amino acid sequence: ISWNSGDI (SEQ ID NO: 86); and CDR-H3 containing the amino acid sequence: AKDTLSGTG TTWYYFDY (SEQ ID NO: 88); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QGISSY (SEQ ID NO: 92); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 94); and CDR-L3 containing the amino acid sequence: QQVNSYPLT (SEQ ID NO: 96); (7) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 100); CDR-H2 containing the amino acid sequence: ISYDGSNK (SEQ ID NO: 102); and CDR-H3 containing the amino acid sequence: AQDGSSAIYYFYGMDV (SEQ ID NO: 104); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QDINNY (SEQ ID NO: 108); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 110); and CDR-L3 containing the amino acid sequence: LQYNSYHPT (SEQ ID NO: 112); (8) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 116); CDR-H2 containing the amino acid sequence: IWYDGSNK (SEQ ID NO: 118); and CDR-H3 containing the amino acid sequence: ARGEHYYGSGPFDP (SEQ ID NO: 120); and light chain variable region

- 4 043756 immunoglobulin, which contains: CDR-L1 containing the amino acid sequence: QSISNY (SEQ ID NO: 124); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 126); and CDR-L3 containing the amino acid sequence: QQSYSSPLT (SEQ ID NO: 128); (9) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GGSISSFGYY (SEQ ID NO: 132); CDR-H2 containing the amino acid sequence: IYYSGSI (SEQ ID NO: 134); and CDR-H3 containing the amino acid sequence: ARERDYGDYFDY (SEQ ID NO: 136); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSVSSN (SEQ ID NO: 140); CDR-L2 containing the amino acid sequence: GAS (SEQ ID NO: 142); and CDR-L3 containing the amino acid sequence: QQYNNWPLT (SEQ ID NO: 144); (10) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 148); CDR-H2 containing the amino acid sequence: IWYDGSNK (SEQ ID NO: 150); and CDR-H3 containing the amino acid sequence: ARDQDYYGSGSSYGMDV (SEQ ID NO: 152); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 156); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 158); and CDR-L3 containing the amino acid sequence: QQSYSTPLT (SEQ ID NO: 160); (11) an immunoglobulin heavy chain variable region, which contains a CDR-H1 containing the amino acid sequence: GFTFSTYG (SEQ ID NO: 164); CDR-H2 containing the amino acid sequence: IWYDGTNK (SEQ ID NO: 166); and CDR-H3 containing the amino acid sequence: ARDPSGGDHYYYYGMDV (SEQ ID NO: 168); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QDISNY (SEQ ID NO: 172); CDR-L2 containing the amino acid sequence: DAS (SEQ ID NO: 174); and CDR-L3 containing the amino acid sequence: QQYDNIPIT (SEQ ID NO: 176); (12) an immunoglobulin heavy chain variable region, which contains a CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 180); CDR-H2 containing the amino acid sequence: ISFDERNK (SEQ ID NO: 182); and CDR-H3 containing the amino acid sequence: ASEVGYSFGHDAFDI (SEQ ID NO: 184); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QDISNY (SEQ ID NO: 188); CDR-L2 containing the amino acid sequence: DAS (SEQ ID NO: 190); and CDR-L3 containing the amino acid sequence: QQYDNFPLT (SEQ ID NO: 192); (13) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFTFNNYA (SEQ ID NO: 196); CDR-H2 containing the amino acid sequence: ISGSGDST (SEQ ID NO: 198); and CDR-H3 containing the amino acid sequence: AKDQGLYYYGSGSFDY (SEQ ID NO: 200); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 204); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 206); and CDR-L3 containing the amino acid sequence: QQSYSTPFT (SEQ ID NO: 208); (14) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFAFSDSA (SEQ ID NO: 212); CDR-H2 containing the amino acid sequence: IRNKANRFAT (SEQ ID NO: 214); and CDR-H3 containing the amino acid sequence: ARHGHDTLTEGYGMDV (SEQ ID NO: 216); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (15) an immunoglobulin heavy chain variable region, which contains a CDR-H1 containing the amino acid sequence: GGTFSSYT (SEQ ID NO: 228); CDR-H2 containing the amino acid sequence: IIPLYGTA (SEQ ID NO: 230); and CDR-H3 containing the amino acid sequence: ASTLELRAFDAFDI (SEQ ID NO: 232); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (16) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GGSISSGGYY (SEQ ID NO: 236); CDR-H2 containing the amino acid sequence: IYYSGST (SEQ ID NO: 238); and CDR-H3 containing the amino acid sequence: ARAPPYNWFDY (SEQ ID NO: 240); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (17) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFTFSDYY (SEQ ID NO: 244); CDR-H2 containing the amino acid sequence: ISNSGNTQ (SEQ ID NO: 246); and CDR-H3 containing amino acids

- 5 043756 new sequence: TREGLEYSSSEPFDY (SEQ ID NO: 248); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (18) an immunoglobulin heavy chain variable region, which contains a CDR-H1 containing the amino acid sequence: GYTFTAYY (SEQ ID NO: 252); CDR-H2 containing the amino acid sequence: INPNNGDT (SEQ ID NO: 254); and CDR-H3 containing the amino acid sequence: ARDDLAAAGIGWFDS (SEQ ID NO: 256); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (19) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFTFDDYA (SEQ ID NO: 260); CDR-H2 containing the amino acid sequence: ISWNSESI (SEQ ID NO: 262); and CDR-H3 containing the amino acid sequence: AKAPYSGTYFEYFR H (SEQ ID NO: 264); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (20) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFTFSSYG (SEQ ID NO: 268); CDR-H2 containing the amino acid sequence: ISYDGSNK (SEQ ID NO: 270); and CDR-H3 containing the amino acid sequence: AKDDWNYDAFDI (SEQ ID NO: 272); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSI SSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (21) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GGSISSSGYY (SEQ ID NO: 276); CDR-H2 containing the amino acid sequence: IYYSGST (SEQ ID NO: 278); and CDR-H3 containing the amino acid sequence: ARVDYGSGSSFDY (SEQ ID NO: 280); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (22) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GYTFTSYG (SEQ ID NO: 284); CDR-H2 containing the amino acid sequence: ISGFNGRT (SEQ ID NO: 286); and CDR-H3 containing the amino acid sequence: ARDGLEKLGDY (SEQ ID NO: 288); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (23) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFTFSNSG (SEQ ID NO: 292); CDR-H2 containing the amino acid sequence: IWHDGSYK (SEQ ID NO: 294); and CDR-H3 containing the amino acid sequence: ARDDYYASGTSVDV (SEQ ID NO: 296); and an immunoglobulin light chain variable region, which contains CDR-L1 containing the amino acid sequence: QSISSY (SEQ ID NO: 220); CDR-L2 containing the amino acid sequence: AAS (SEQ ID NO: 222); and CDR-L3 containing the amino acid sequence: QQSYSTPPIT (SEQ ID NO: 224); (24) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GYTFTGYY (SEQ ID NO: 300); CDR-H2 containing the amino acid sequence: INPNSGGT (SEQ ID NO: 302); and CDR-H3 containing the amino acid sequence: AREEVDDFWSGYLDY (SEQ ID NO: 304); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSVSSSY (SEQ ID NO: 308); CDR-L2 containing the amino acid sequence: GAS (SEQ ID NO: 310); and CDR-L3 containing the amino acid sequence: QQYGSSPWT (SEQ ID NO: 312); (25) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GFAVNGDY (SEQ ID NO: 316); CDR-H2 containing the amino acid sequence: IYSSGNT (SEQ ID NO: 318); and CDR-H3 containing the amino acid sequence: ARDFPPMSGADY (SEQ ID NO: 320); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSVSSSY (SEQ ID NO: 308); CDR-L2 containing the amino acid sequence: GAS (SEQ ID NO: 310); and CDR-L3 containing the amino acid sequence: QQYGSSPWT (SEQ ID NO: 312); (26) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GYTLTELS (SEQ ID NO: 324); CDR-H2 containing amino acid sequence

- 6 043756 activity: FDPEHGKI (SEQ ID NO: 326); and CDR-H3 containing the amino acid sequence: ATFYNWNSYYFGMDV (SEQ ID NO: 328); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSVSSSY (SEQ ID NO: 308); CDR-L2 containing the amino acid sequence: GAS (SEQ ID NO: 310); and CDR-L3 containing the amino acid sequence: QQYGSSPWT (SEQ ID NO: 312); (27) an immunoglobulin heavy chain variable region, which contains a CDR-H1 containing the amino acid sequence: GFTFSSYA (SEQ ID NO: 332); CDR-H2 containing the amino acid sequence: VSGSADIT (SEQ ID NO: 334); and CDR-H3 containing the amino acid sequence: AKDKVYNWNYGIYYGMDV (SEQ ID NO: 336); and an immunoglobulin light chain variable region, which contains CDR-L1 containing the amino acid sequence: QSVSSSY (SEQ ID NO: 308); CDR-L2 containing the amino acid sequence: GAS (SEQ ID NO: 310); and CDR-L3 containing the amino acid sequence: QQYGSSPWT (SEQ ID NO: 312); (28) an immunoglobulin heavy chain variable region, which contains: CDR-H1 containing the amino acid sequence: GGSISSSSYY (SEQ ID NO: 340); CDR-H2 containing the amino acid sequence: IYYSGST (SEQ ID NO: 342); and CDR-H3 containing the amino acid sequence: ARQGRWERENFDY (SEQ ID NO: 344); and an immunoglobulin light chain variable region, which contains CDR-L1 containing the amino acid sequence: QSVSSSY (SEQ ID NO: 308); CDR-L2 containing the amino acid sequence: GAS (SEQ ID NO: 310); and CDR-L3 containing the amino acid sequence: QQYGSSPWT (SEQ ID NO: 312); and/or (29) an immunoglobulin heavy chain variable region, which contains a CDR-H1 containing the amino acid sequence: DESFSDYY (SEQ ID NO: 348); CDR-H2 containing the amino acid sequence: ITHSGST (SEQ ID NO: 350); and CDR-H3 containing the amino acid sequence: ARGGDYGGLLDY (SEQ ID NO: 352); and an immunoglobulin light chain variable region, which contains: CDR-L1 containing the amino acid sequence: QSVSSSY (SEQ ID NO: 308); CDR-L2 containing the amino acid sequence: GAS (SEQ ID NO: 310); and CDR-L3 containing the amino acid sequence: QQYGSSPWT (SEQ ID NO: 312). For example, in an embodiment of the present invention, the antigen binding protein (e.g., an antibody or an antigen binding fragment thereof) comprises (a) an immunoglobulin heavy chain that contains the amino acid sequence of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354; and/or (b) an immunoglobulin light chain that contains the amino acid sequence of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, or 306. In an embodiment In embodiments of the present invention, the antigen binding protein is multispecific (eg, bispecific, multiparatopic, or biparatopic).

In an embodiment of the present invention, the antigen binding protein (eg, an antibody or an antigen binding fragment thereof) comprises one or more of the following properties: inhibits the growth of Plasmodium falciparum in human red blood cells; inhibits the growth of Plasmodium falciparum strains D10, Dd2, 7G8, W2-mef, 3D7, HB3, FCR-1/FVO, Cam3.II or RF7 in human erythrocytes; blocks the binding of the PfRH5 polypeptide to the basigin polypeptide; for example, at a concentration of about 6.67 microM, it causes maximal growth inhibition (e.g., in vitro) of Plasmodium falciparum (e.g., strain FCR-1/FVO) in heat-inactivated human or Aotus monkey serum (e.g., as measured by lactate dehydrogenase (LDH) activity ) parasite), which is about 1-10% (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%) (relative to uninfected red blood cells) higher than in non-heat-inactivated serum human or monkey Aotus, respectively; when exposed to the specified antigen-binding protein does not cause mutation of PfRH5 in Plasmodium falciparum (for example, strain 3D7), for example, in vitro after about 45 days of gradual increase in antibody concentration, for example, from about 1XEC ₅₀ to about 110XEC ₅₀ ; and/or binds to cPfRH5, which does not contain the amino terminal residues M1-Y139 and includes residues K140-Q526, but does not contain K247L295 and has, for example, mutations T216A and T299A, where the antigen binding protein contains the amino acid sequence herein.

The invention also includes a complex comprising an antigen binding protein (eg, an antibody or an antigen binding fragment thereof) linked to a Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) polypeptide. For example, PfRH5 is located on the surface of a cell, such as Plasmodium falciparum (eg, Plasmodium falciparum merozoites), for example, in a subject (eg, human). In one embodiment of this invention, PfRH5 is located on the surface of Plasmodium falciparum, for example, a merozoite in a red blood cell.

This invention also provides a method for producing an anti-PfRH5 antigen binding protein (e.g., an antibody or an antigen binding fragment thereof) set forth herein, or an immunoglobulin chain thereof, comprising: (a) introducing one or more polynucleotides encoding the immunoglobulin chain of said antigen binding protein into a host cell (eg, a Chinese hamster ovary cell); (b) culturing the host cell under conditions favorable for expression of the polynucleotide; and (c) optionally isolating an antigen binding protein, or

- 7 043756 immunoglobulin chain from the host cell and/or the environment in which the host cell is grown. The antigen binding protein or immunoglobulin chain, which is a product of such a method, also forms part of the present invention.

An immunochromatographic antigen test strip containing an anti-PfRH5 antigen binding protein as set forth herein (eg, an antibody or an antigen binding fragment thereof) is part of the present invention. Methods for detecting Plasmodium falciparum in a blood sample from a subject and/or from a subject's body using a test strip are also part of the present invention.

A polypeptide comprising: (a) CDR-H1, CDR-H2 and CDR-H3 of the immunoglobulin heavy chain variable region of an immunoglobulin chain, which contains the amino acid sequence SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354; or (b) CDR-L1, CDR-L2 and CDR-L3 of the immunoglobulin light chain variable region of an immunoglobulin chain which contains the amino acid sequence of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154 , 170, 186, 202, 218 or 306 are also part of this invention. Polynucleotides (eg, DNA or RNA) encoding such a polypeptide are also part of the present invention, along with a vector that contains the polynucleotide.

The present invention also provides a host cell (eg, a Chinese hamster ovary cell) containing an antigen binding protein (eg, an antibody or an antigen binding fragment thereof) or an immunoglobulin chain or polypeptide or polynucleotide or vector as set forth herein.

The present invention also provides a composition or kit that contains one or more (e.g., 1, 2, 3 or 4) anti-PfRH5 antigen binding proteins (e.g., an antibody or antigen binding fragment thereof) set forth herein, optionally in combination with additional a therapeutic agent (eg, an antiparasitic drug, chloroquine, atovaquone, proguanil, artemether, lumefantrine, mefloquine, quinine, quinidine, doxycycline (optionally in combination with quinine), clindamycin, vaccine, antimalarial vaccine or RTS, S/AS01). The present invention also provides a pharmaceutical composition comprising the anti-PfRH5 antigen binding protein set forth herein and a pharmaceutically acceptable carrier and optionally an additional therapeutic agent.

The present invention also provides a vessel or injection device (eg, an auto-injector or prefilled syringe) containing an anti-PfRH5 antigen binding protein (eg, an antibody or antigen binding fragment thereof) or composition (eg, a pharmaceutical composition) set forth herein.

The present invention provides a method of treating or preventing Plasmodium falciparum infection (e.g., malaria) in a subject (e.g., human) in need thereof, comprising administering (e.g., parenterally) a therapeutically effective amount of an anti-PfRH5 antigen-binding protein discussed herein, optionally in combination with an additional therapeutic agent. In an embodiment of the invention, the subject is diagnosed as suffering from a Plasmodium falciparum infection (eg, malaria) before treatment is initiated. For example, in an embodiment of the present invention, a subject is diagnosed using a chromatographic test strip as set forth herein. In one embodiment of the present invention, the subject is not infected with Plasmodium falciparum, but is administered a therapeutically effective amount of anti-PfRH5 antigen binding protein prophylactically, i.e. to prevent such infection.

The present invention also provides a method for diagnosing Plasmodium falciparum infection in a subject, comprising contacting an anti-PfRH5 antigen binding protein of the present invention with a sample (eg, blood) from the subject and, if a complex between the antigen binding protein and a PfRH5 polypeptide is detected in the sample, determining that the subject is infected with Plasmodium falciparum. For example, the complex may be formed on a chromatographic assay test strip as set forth herein containing an anti-PfRH5 antigen binding protein of the invention and a PfRH5 polypeptide (from a subject sample).

The present invention provides a method of administering an anti-PfRH5 antigen binding protein (e.g., an antibody or an antigen binding fragment thereof) set forth herein to a subject (e.g., a human), comprising injecting the antigen binding protein into the subject's body, optionally in combination with an additional therapeutic agent , for example, subcutaneously, intravenously or intramuscularly.

This invention also covers any immunoglobulin polypeptide or polynucleotide set forth herein, for example, containing any amino acid sequence of a member selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20 , 22, 24, 26, 28, 30, 34, 36, 38, 40, 42, 44, 46, 50, 52, 54, 56, 58, 60, 62, 66, 68, 70, 72, 74, 76 , 78, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104, 106, 108, 110, 114, 116, 118, 120, 122, 124, 126, 130, 132, 134 , 136, 138, 140, 142, 146, 148, 150,

152, 154, 156, 158, 162, 164, 166, 168, 170, 172, 174, 178, 180, 182, 184, 186, 188, 190, 194, 196, 198, 200,

202, 204, 206, 210, 212, 214, 216, 218, 220, 222, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,

- 8 043756

250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 2 98, 300, 302, 304, 306, 308, 310, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 3 50, 352, 354, 356, 358 and 360; or a polynucleotide containing any nucleotide sequence from a member selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 17, 19, 21, 23, 25, 27, 29, 33, 35 , 37, 39, 41, 43, 45, 49, 51, 53, 55, 57, 59, 61, 65, 67, 69, 71, 73, 75, 77, 81, 83, 85, 87, 89, 91 , 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 129, 131, 133, 135, 137, 139, 141, 145, 147, 14 9 , 151, 153, 155, 157, 161, 163, 165, 167, 169, 171, 173, 177, 179, 181, 183, 185, 187, 189,

193, 195, 197, 199, 201, 203, 205, 209, 211, 213, 215, 217, 219, 221, 225, 227, 229, 231, 233, 235, 237,239,

241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281,283,

285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 313, 315, 317, 319, 321, 323, 325, 327,329,

331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357 and 359.

Brief description of graphic materials

The drawing shows the alignment of PfRH5 sequences corresponding to each PfRH5-specific antibody, 45 days after gradually increasing the antibody pressure (from 1 χ EC50 to 110 χ EC50).

Detailed description of the invention

Plasmodium falciparum is a protozoan parasite, one of the Plasmodium species that causes malaria in humans, which can be transmitted by female Anopheles mosquitoes. The malaria caused by this species (which may be called malignant tertian malaria) is a very dangerous form of malaria with a high morbidity and mortality rate. See, for example, Gardner et al., Genome sequence of the human malaria parasite Plasmodium falciparum, Nature 419(6906): 498511 (2002). Plasmodium falciparum includes any strain thereof that exhibits sensitivity to the antigen binding protein of this invention, for example D10, Dd2, 7G8, W2-mef, 3D7, HB3, FCR-1/FVO, Cam3.II or RF7. Plasmodium falciparum infection refers to the invasion and proliferation of Plasmodium falciparum in a subject's body. The present invention provides various antigen binding proteins that are effective for the treatment or prevention of Plasmodium falciparum infection.

An anti-PfRH5 antigen binding protein is a single polypeptide (e.g., ScFv (single chain variable fragment)) or a complex of more than one polypeptide (e.g., a tetrameric IgG antibody) that specifically binds to a PfRH5 polypeptide, e.g., an anti-PfRH5 antibody or antigen-binding fragment , regardless of whether they are monospecific or multispecific (eg, bispecific), monovalent or multivalent (eg, bivalent). A monovalent antigen binding protein has one antigen binding domain, while a bivalent antigen binding protein has two antigen binding domains.

Basigin (BSG, extracellular matrix metalloproteinase inducer, EMMPRIN, CD147) is a polypeptide that is a target on red blood cells to which PfRH5 binds. In an embodiment of the present invention, the amino acid sequence of basigin is set forth under Uniprot accession number Q54A51. See, for example, Crosnier et al, Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum, Nature. 2011 Nov 9; 480(7378):534-7. In one embodiment of the present invention, the antigen binding protein of the present invention blocks the binding between PfRH5 and BSG.

Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5)

Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5) is a member of a superfamily of erythrocyte ligands called reticulocyte binding-like proteins (RBLs). Evidence suggests that PfRH5 is required for the spread of Plasmodium falciparum infection to the bloodstream stage. PfRH5 binds erythrocytes and is involved in species tropism during erythrocyte invasion. See, for example, Bustamante et al., Vaccine. 2013 Jan 2; 31(2):373-379.

In an embodiment of the present invention, PfRH5 contains the amino acid sequence:

- 9 043756

MIRIKKKLIL TIIYIHLFIL NRLSFENAIK KTKNQENNLT LLPIKSTEEE KDDIKNGKDI

KKEIDNDKEN IKTNNAKDHS TYIKSYLNTN VNDGLKYLFI PSHNSFIKKY SVFNQINDGM LLNEKNDVKN NEDYKNVDYK NVNFLQYHFK ELSNYNIANS IDILQEKEGH LDFVIIPHYT FLDYYKHLSY NSIYHKSSTY GKCIAVDAFI KKINETYDKV I<SI<CNDII<ND LIATIKKLEH PYDINKNDD SYRYDISEEI DDKSEETDDE TEEVEDSIQD TDSNHTPSNK KKNDLMNRTF KKMMDEYNTK KKKLIKCIKN HENDFNKICM DMKNYGTNLF EQLSCYNNNF CNTNGIRYHY DEYIHKLILS VKSKNLNKDL SDMTNILQQS ELLLTNLNKK MGSYIYIDTI

KFIHKEMKHI

FNRIEYHTKIINDKTKIIQD KIKLNIWRTF QKDELLKRIL DMSNEYSLFI TSDHLRQMLY NTFYSKEKHL NNIFHHLIYV LQMKFNDVPI KMEYFQTYKK NKPLTQ (SEQ ID NO: 361)

Polypeptide PfRH5ANL.6his (PfRH5 (K140-Q526; del M1-Y139; del K247-L295; T216A; T299A)), which lacks N-terminal residues 1-139 and residues 247-295 and has mutations T216A and T299A, and also the C-terminal His ₆ tag is part of this invention together with the polynucleotides encoding the polypeptide. In an embodiment of the invention, the PfRH5ANL.6his polypeptide contains the amino acid sequence of SEQ ID NO: 362:

KNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYH KSSTYGKYIAVDAFIKKINEAYDKVKSKCNDIKNDLIATIKKLEHPYDINNMNRAFKKM MDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYD EYIHKLILSVKS KNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFN RIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFY SKEKHLNNIFHHLIYVLQMKFNDVPII<MEYFQTYI<KNI<PLTQHHHHHH

In an embodiment of the present invention, the polypeptide is in crystallized form or in non-crystallized form.

Antibodies and antigen-binding fragments

The present invention provides antigen binding proteins, such as antibodies and antigen binding fragments thereof, that specifically bind to the PfRH5 protein or an antigenic fragment thereof. The immunoglobulin chains of this invention are described herein in Example 1 in Table. 1-1.

The term antibody as used herein refers to immunoglobulin molecules containing four polypeptide chains, two heavy chains (HC) and two light chains (LC), linked together by disulfide bonds (i.e., full-length antibody molecules), and their multimers (for example, IgM), for example, H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; N1N29209R2, N1N29214R2; or N1N29215R2. Each heavy chain (HC) contains a heavy chain variable region (HCVR or VH) (e.g. SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210 , 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354) and the heavy chain constant region (consisting of the _CH1 , CH2 and _CH2 domains 3). Each light chain (LC) consists of a light chain variable region (LCVR or VL) (for example, SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218 or 306) and the light chain constant region (CL). The V _H and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are separated by regions that are more conserved called framework regions (FRs). Each VH and _VL contains three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the present invention, the FR antibody (or antigen binding fragment thereof) is identical to human germline sequences or may be naturally or artificially modified.

Typically, the variable domains of both the heavy and light chains of an immunoglobulin contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). Typically, from N-terminus to C-terminus, the variable domains of both the light and heavy chains contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In an embodiment of the present invention, the assignment of amino acids to each domain follows the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 ^th ed.; NIHPubl. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

- 10 043756

The present invention provides monoclonal anti-PfRH5 antigen-binding proteins, for example, antibodies and antigen-binding fragments thereof, as well as monoclonal compositions containing a plurality of isolated monoclonal antigen-binding proteins. The term monoclonal antibody as used herein refers to a population of essentially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minute quantities. The plurality of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., as discussed above, in amino acid sequence, excluding possible naturally occurring mutations that may be present in minor quantities) antibodies and fragments that is higher than that which usually found in nature, such as in the blood of a host organism such as a mouse or human.

In an embodiment of the present invention, the anti-PfRH5 antigen binding protein, e.g., antibody or antigen binding fragment, comprises a heavy chain constant domain, e.g., of the IgA type (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4) or IgM. In an embodiment of the present invention, the antigen binding protein, eg, antibody or antigen binding fragment, comprises a light chain constant domain, eg of the kappa or lambda type.

The term human antigen binding protein, such as an antibody or antigen binding fragment, as used herein, includes antibodies and fragments having variable and constant regions derived from human germline immunoglobulin sequences, whether in a human cell or grafted into a non-human cell, for example , mouse cage. See, for example, US 8502018, US 6596541 or US 5789215. Human monoclonal antibodies (mAbs) of this invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example in the CDR and, in particular, in CDR3. However, the term human antibody as used herein is not intended to include mAbs in which CDR sequences derived from the germ line of other mammalian species (eg, mouse) have been grafted onto human FR sequences. The term includes antibodies produced recombinantly in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies isolated or generated from a human subject.

This invention includes anti-PfRH5 chimeric antigen binding proteins, eg antibodies and antigen binding fragments thereof, and methods of using them. As used herein, a chimeric antibody is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies are of different species (see, for example, US4816567; and Morrison et al., (1984) Proc. Natl Acad Sci USA 81:6851-6855).

The term recombinant antigen binding proteins, such as antibodies or antigen binding fragments thereof, refers to such molecules generated, expressed, isolated or produced using technologies or methods known in the art, such as recombinant DNA technology, which includes, for example, splicing DNA and transgene expression. The term includes antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice) or in a cellular (e.g., CHO cell) expression system, or isolated from a combinatorial library of recombinant human antibodies.

Recombinant anti-PfRH5 antigen binding proteins, such as the antibodies and antigen binding fragments disclosed herein, can also be produced in the E. coli/T7' expression system. In this embodiment, nucleic acids encoding anti-PfRH5 antibody immunoglobulin molecules of the present invention (e.g., H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29125P; H1H29127P; H1H291 31Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; N1N29196R2; N1N29198R2; N 1H29207P2; H1H29209P2, H1H29214P2 or H1H29215P2) can be inserted into a pET-based plasmid and expressed in the E. coli/T7' system. For example, the present invention includes methods for expressing an antibody or an antigen binding fragment thereof or an immunoglobulin chain thereof in a host cell (e.g., a bacterial host cell such as E. coli, such as BL21 or BL21DE3), comprising expressing T7 RNA polymerase in the cell, which also contains a polynucleotide encoding an immunoglobulin chain that is operably linked to the T7 promoter. For example, in an embodiment of the present invention, a bacterial host cell, such as E. coli, contains a polynucleotide encoding a T7 RNA polymerase gene operably linked to the lac promoter, and expression of the polymerase and chain is induced by incubating the host cell with IPTG (isopropyl beta -O-thiogalactopyranoside). See US4952496 and US5693489 or Studier & Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986 May

- 11 043756

5;189(1): 113-30.

There are several methods for producing recombinant antibodies that are known in the art. One example of a method for recombinant production of antibodies is disclosed in US 4,816,567.

Transformation can be carried out by any known method of introducing polynucleotides into the host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, liposome encapsulation of the polynucleotide(s), biolistic injection, and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells using viral vectors. Methods for transforming cells are well known in the art. See, for example, US Pat. Nos. 4,399,216; 4912040; 4740461 and 4959455.

Polynucleotides encoding immunoglobulins set forth herein (e.g., containing the nucleotide sequence SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 17, 19, 21, 23, 25, 27, 29, 33, 35 , 37, 39, 41, 43, 45, 49, 51, 53, 55, 57, 59, 61, 65, 67, 69, 71, 73, 75, 77, 81, 83, 85, 87, 89, 91 , 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 125, 129, 131, 133, 135, 137, 139, 141, 145, 147, 14 9 , 151,

153, 155, 157, 161, 163, 165, 167, 169, 171, 173, 177, 179, 181, 183, 185, 187, 189, 193, 195, 197, 199,201,

203, 205, 209, 211, 213, 215, 217, 219, 221, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249,

251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291,293,

295, 297, 299, 301, 303, 305, 307, 309, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337,339,

341, 343, 345, 347, 349, 351, 353, 355, 357 and 359), which are contained in a vector and/or are operably linked to an expression control sequence such as a promoter, form part of the present invention. The promoter may be, for example, a CMV promoter (eg, the human cytomegalovirus (CMV) major immediate-early (MIE) promoter or a mouse CMV promoter) or an SV40 promoter (eg, the SV40 early promoter). The vector may be a plasmid (eg, a circular plasmid or a linearized plasmid) or a viral vector that can be ectopically maintained in a host cell or integrated into the host chromosome. Such host cells form part of the present invention.

A polynucleotide is operably linked when it is operably linked to another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence. Typically, although not always, operably linked means that the DNA sequences being linked are contiguous, and in the case of a secretory leader sequence, contiguous and in reading phase. At the same time, enhancers do not have to be adjacent.

Thus, the present invention includes recombinant methods for producing an anti-PfRH5 antigen binding protein, such as an antibody or antigen binding fragment thereof of the present invention, or an immunoglobulin chain thereof, comprising (i) introducing one or more polynucleotides (for example, including a nucleotide sequence in any one or more of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345 and 353) , encoding light and/or heavy immunoglobulin chains of an antigen-binding protein, for example, H1H29O89P; Н1Н29О94Р; N1N291OOR; Н1Н291О4Р; Н1Н291О6Р; Н1Н291О9Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н292О7Р2; H1H292O9P2, H1H29214P2 or H1H29215P2, for example, where the polynucleotide is in the vector; and/or integrated into the host cell chromosome and/or operably linked to a promoter; (ii) culturing a host cell (eg, CHO or Pichia or Pichia pastoris) under conditions favorable for expression of the polynucleotide, and (iii) optionally isolating the antigen binding protein (eg, antibody or fragment) or chain from the host cell and/or medium , in which the host cell is grown. When creating an antigen-binding protein (e.g., an antibody or antigen-binding fragment) containing more than one immunoglobulin chain, for example, an antibody that contains two immunoglobulin heavy chains and two immunoglobulin light chains, coexpression of the chains in the same host cell results in association of the chains, e.g. cell or on the surface of a cell or extracellularly if such chains are secreted so as to form an antigen-binding protein (eg, an antibody or antigen-binding fragment). The methods of this invention include those in which only the immunoglobulin heavy chain or only the immunoglobulin light chain (eg, any of those discussed herein, including mature fragments and/or variable domains thereof) are expressed in a cell. Such single chains are useful, for example, as mediators in the expression of an antibody or antigen-binding fragment that contains such a chain. For example, the present invention also includes anti-PfRH5 antigen-binding proteins, such as antibodies and antigen-binding fragments thereof containing an immunoglobulin heavy chain (or a variable domain thereof or containing

- 12 043756 its CDR), encoded by a polynucleotide containing the nucleotide sequences set forth in SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 233, 241, 249, 257, 265, 273, 281, 289, 297, 313, 321, 329, 337, 345, or 353, and an immunoglobulin light chain (or a variable domain thereof or CDRs containing it) encoded by the nucleotide sequence set forth in SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217 or 305, which are the product of such preparation methods, and optionally, the purification methods set forth in this document. For example, in an embodiment of the present invention, the product of the method is an anti-PfRH5 antigen binding protein, which is an antibody or fragment containing a VH containing the amino acid sequence SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354, and VL containing amino acid sequence SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218 or 306.

In an embodiment of the present invention, a method of creating an anti-PfRH5 antigen binding protein, eg, an antibody or an antigen binding fragment thereof, includes a method of purifying the antigen binding protein, eg, by column chromatography, precipitation, and/or filtration. The product of such a process also forms part of the present invention.

Eukaryotic and prokaryotic host cells, including mammalian cells, can be used as hosts for the expression of an anti-PfRH5 antigen binding protein (eg, an antibody or an antigen binding fragment thereof). Such host cells are well known in the art, and many are available from the American Type Culture Collection (ATCC). These host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (eg, Hep G2) , A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, equine and hamster cells. Other cell lines that can be used are insect cell lines (eg Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include cells of yeast and filamentous fungi, including, for example, Pichia, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenulapolymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Asper Gillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrellapatens and Neurospora crassa. The present invention includes an isolated host cell (eg, a CHO cell) containing an antigen binding protein such as H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; N1N29209R2, N1N29214R2 or N1N29215R2; or one or more polynucleotides encoding an immunoglobulin chain or chains thereof.

The present invention also includes a Plasmodium falciparum cell that expresses PfRH5, which is bound by an antigen binding protein of the present invention, for example, H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; H1H29209P2, H1H29214P2, or H1H29215P2, for example, where the cell is in the subject's body or is in vitro.

The term specifically binds refers to those antigen-binding proteins (eg, mAbs) having a binding affinity for an antigen, such as the PfRH5 protein (eg, PfR.H5ANL.6his), expressed as a K _D of at least about 10 ^-8 M ( e.g., any K _D specified in Table 5-1 or 5-2 herein) as measured in a real-time marker-free biolayer interferometry assay, e.g., at 25°C or 37°C, e.g., using an Octet® biosensor HTX, or by surface plasmon resonance, such as BIACORE™, or by ELISA (enzyme-linked immunosorbent assay). The present invention includes antigen binding proteins that specifically bind to the PfRH5 protein. In an embodiment of the present invention, the antigen binding protein has K _a , K _d and/or t _1/2 as set out in table. 5-1 or 5-2 in this document.

The terms antigen binding portion of an antibody or antigen binding fragment of an antibody, etc., as used herein, include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein,

- 13 043756 which specifically binds the antigen to form a complex. Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) P(ab') ₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments and (vii) peptides with limited conformational freedom FR3-CDR3-FR4 (eg, containing FR3, FR4 and CDR-H3 or CDR-L3, as set forth herein). Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deletion antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, and small modular immunopharmaceuticals (SMIPs) are also covered by the expression antigen-binding fragment as used herein. document. In an embodiment of the present invention, the antigen binding fragment comprises three or more CDRs from H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; N1N29209R2, N1N29214R2; or H1H29215P2 (eg, CDR-H1, CDR-H2 and CDR-H3; and/or CDR-L1, CDR-L2 and CDR-L3).

In an embodiment, the antigen binding fragment of an antibody will typically comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is adjacent or in frame with one or more framework sequences. In antigen binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be located relative to each other in any suitable relationship. For example, the variable region may be dimeric and contain V _H - VH -, V _H - V _L - or V _L - V _L -dimers. Alternatively, the antigen binding fragment of the antibody may comprise a monomeric VH or VL domain.

In some embodiments, the antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen binding fragment of an antibody of the present invention include: (i) Vh-Ch1; (ii) Vh-Ch2; (iii) Vh-Ch3; (iv) Vh-Ch1-Ch2; (v) Vh-Ch1-Ch2-Ch3; (vi) Vh-Ch2Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) VlC _h 2-C _h 3; and (xiv) V _l -C _l . In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may either be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen binding fragment of an antibody of the present invention may contain a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric VH- or _VL- domains (eg via disulfide bond(s)).

Antigen-binding proteins (eg, antibodies and antigen-binding fragments) can be monospecific or multispecific (eg, bispecific). Multispecific antigen binding proteins are discussed further herein.

In an embodiment of the present invention, the antigen binding proteins of the present invention (e.g., an antibody or antibody fragment) can be conjugated to a fragment, such as a therapeutic fragment (immunoconjugate), such as an antimalarial drug, a second anti-PfRH5 antibody, or any other therapeutic fragment useful for treating an infection Plasmodium falciparum (See, for example, below).

This invention also provides a complex containing an anti-PfRH5 antigen binding protein, such as an antibody or antigen binding fragment discussed herein, in complex with a PfRH5 polypeptide or antigenic fragment thereof and/or a secondary antibody or antigen binding fragment thereof (e.g., a detectably labeled secondary antibody) that specifically binds to an anti-PfRH5 antibody or fragment. In an embodiment of the present invention, the complex is in vitro (eg, immobilized on a solid substrate) or in the body of a subject. In an embodiment of the present invention, PfRH5 is immobilized on a solid substrate (eg, a chromatography test strip) or located on the surface of a cell, such as Plasmodium falciparum. Immobilized anti-PfRH5 antibodies and antigen binding fragments thereof that are covalently linked to an insoluble matrix material (eg glass or polysaccharide such as agarose or sepharose, eg a bead or other particle thereof) are also part of the present invention; optionally wherein the immobilized antibody forms a complex with PfRH5 or an antigenic fragment thereof or a secondary antibody or fragment thereof.

Isolated antigen-binding proteins (for example, antibodies or antigen-binding fragments thereof)

- 14 043756 ments), polypeptides, polynucleotides and vectors are at least partially free of other biological molecules from the cells or cell culture from which they are derived. Such biological molecules include nucleic acids, proteins, other antibodies or antigen binding fragments, lipids, carbohydrates or other material such as cell debris and growth media. The isolated antigen binding protein may further be at least partially free of components of the expression system, such as biological molecules from the host cell or its growth medium. Generally, the term isolated is not intended to denote the complete absence of such biological molecules or the absence of water, buffers or salts, or components of a pharmaceutical formulation that contains antigen-binding proteins (eg, antibodies or antigen-binding fragments).

This invention includes antigen-binding proteins, for example, antibodies or antigen-binding fragments that bind to the same epitope as the antigen-binding protein of this invention (for example, H1H29089R; H1H29094R; H1H29100R; H1H29104R; H1H29106R; H1H29109R; H1H29125R; H1H29 127Р;Н1Н29131Р;Н1Н29134Р; N1N29138R; N1N29141R; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2: N1N2 9183Р2: Н1Н29187Р2: Н1Н29192Р2: Н1Н29196Р2: Н1Н29198Р2: Н1Н29207Р2; Н1Н29209Р2, Н1Н29214Р2 or Н1Н29215Р2). For example, the invention includes antigen-binding proteins that bind to an epitope of a PfRH5 variant (e.g., PfRH5ANL.6his) lacking amino acids M1-Y139 and containing residues K140-Q526, but lacking K247-L295 and having mutations T216A and T299A (optionally in including the HisX6 tag).

The term epitope refers to an antigenic determinant (eg, on a PfRH5 polypeptide) that interacts with a specific antigen-binding site of an antigen-binding protein, eg, a variable region of an antibody molecule known as a paratope. One antigen can have more than one epitope. Thus, different antibodies may bind to different sites on the antigen and may have different biological effects. The term epitope can also refer to a site on an antigen to which B and/or T cells respond, and/or a region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., consist of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface moieties of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in some embodiments, may have specific three-dimensional structural characteristics and/or specific charge.

Methods for determining the epitope of an antigen binding protein, for example, an antibody or fragment or polypeptide, include alanine mutation scanning assay, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide digestion assay, crystallographic studies, NMR analysis. In addition, methods such as epitope removal, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antigen binding protein (e.g., an antibody or fragment or polypeptide) interacts is hydrogen/deuterium exchange detected by mass spectrometry (See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal Chem 73:256A-265A).

The present invention includes antigen binding proteins that compete for binding to PfRH5, eg, an epitope of a PfRH5 variant as discussed herein, with an antigen binding protein of the present invention, eg, H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; N1N29179R2: N1N29183R2: N1N29187R2: N1N29192R2: N1N29196R2: N1N29198R2: N1N29207R2; N1N29209R2, N1N29214R2 or N1N29215R2. The term compete, as used herein, refers to an antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) that binds to an antigen (e.g., PfRH5 or PfRH5ANL.6his) and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) fragment) with antigen. The term also includes competition between two antigen-binding proteins, eg, antibodies in both orientations, ie, a first antibody that binds and blocks the binding of a second antibody, and vice versa. In certain embodiments, the first antigen binding fragment (eg, antibody) and the second antigen binding fragment (eg, antibody) can bind to the same epitope. Alternatively, the first and second antigen-binding proteins (eg, antibodies) may bind to different but, eg, overlapping epitopes, wherein binding of one inhibits or blocks binding of the second antibody, eg, through steric hindrance. Competition between

- 15 043756 antigen-binding proteins (eg antibodies) can be measured by methods known in the art, for example, by real-time marker-free biolayer interferometry. In an embodiment of the present invention, competition between the first and second anti-PfRH5 antigen binding protein (e.g., antibody) is determined by measuring the ability of the immobilized first anti-PfRH5 antigen binding protein (e.g., antibody) (not initially complexed with the PfRH5 protein) to bind to the PfRH5 protein or its fragment forming a complex with a second anti-PfRH5 antigen-binding protein (for example, an antibody). The reduced ability of the first anti-PfRH5 antigen binding protein (eg, antibody) to bind to the complexed PfRH5 protein compared to the non-complexed PfRH5 protein indicates that the first and second anti-PfRH5 antigen binding proteins (eg, antibodies) are competing. The degree of competition can be expressed as a percentage reduction in binding. Such competition can be measured by real-time, marker-free biolayer interferometry, such as the Octet RED384 biosensor (Pall ForteBio Corp.), by ELISA (enzyme-linked immunosorbent assay) or SPR (surface plasmon resonance).

Competition for binding between anti-PfRH5 antigen-binding proteins (eg, monoclonal antibodies (mAbs)) can be determined by real-time, marker-free biolayer interferometry on the Octet RED384 biosensor (Pall ForteBio Corp.). For example, to determine the competition between two anti-PfRH5 monoclonal antibodies, the anti-PfRH5 mAb can first be captured on Octet biosensor tips coated with anti-hFc antibodies (Pall ForteBio Corp., no. 18-5060) by immersing the tips in an anti-PfRH5 mAb solution (hereinafter referred to as mAb1). As a positive control for blocking, antibody-captured biosensor tips can then be saturated with a known blocking isotype control mAb (hereafter referred to as blocking mAb) by immersion in the blocking mAb solution. To determine whether mAb2 competes with mAb1, the biosensor tips can then be immersed in a complexing solution of the PfRH5 polypeptide and a second anti-PfRH5 mAb (hereafter referred to as mAb2), which have been pre-incubated for some time, and the binding of mAb1 to the PfRH5 polypeptide can be determined. Biosensor tips can be washed in buffer between each experimental step. The binding reaction can be monitored in real time throughout the experiment, and the binding response can be recorded at the end of each step.

In an embodiment of the present invention, a competition assay is performed on a biosensor platform (eg, Octet HTX) where one antibody binds/complexes a PfRH5 polypeptide that has been bound to the sensor tip, and the binding of a second antibody to PfRH5 is then assessed. The ability of the second antibody to bind to the precomplexed PfRH5 polypeptide can be assessed, and if reduced binding is detected (e.g., compared to PfRH5 not complexed with the first antibody) or lack of binding of the second antibody, the first and second antibodies are determined to compete for PfRH5 polypeptide binding. In an embodiment of the present invention, the assay is carried out at 25° C. and a pH of about 7, for example 7.4, for example, in the presence of a buffer (for example, HEPES), salt (for example, NaCl), EDTA, surfactant (for example, Tween- 20) and/or a nonspecific protein (for example, bovine serum albumin). In one embodiment of the present invention, binding to a PfRH5 variant (eg, PfRH5ANL.6his) is assessed in a competition assay, for example, where the variant is PfRH5 lacking amino acids M1-Y139 and containing residues K140-Q526 but lacking K247-L295 and having mutations T216A and T299A (optionally including the HisX6 tag). The HisX6 or His ₆ tag is a tag that contains HNNNNH (SEQ ID NO: 365).

Typically, an antibody or antigen binding fragment of the invention that is modified in any way retains the ability to specifically bind to PfRH5, e.g., retains at least 10% of its binding activity for PfRH5 (relative to the parent antibody) if that activity expressed on a molar basis. Preferably, the antibody or antigen binding fragment of the present invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity for PfRH5 of the parent antibody. It is also contemplated that the antibody or antigen binding fragment of the present invention may contain conservative or non-conservative amino acid substitutions (referred to as conservative variants or functionally conserved variants of the antibody) that do not substantially alter its biological activity.

The anti-PfRH5 antigen binding proteins of this invention may contain immunoglobulin chain variants, the amino acid and nucleotide sequences of which are specifically set forth herein.

A polypeptide variant, such as an immunoglobulin chain (for example, VH, VL, HC or LC H1H29O89P; H1H29O94P; H1H291OOP; H1H291O4P; H1H291O6P; H1H291O9P; H1B29125P; H1H29127P; H1H29131P; H1H291 34R; N1N29138R; N1N29141R; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2 ; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2;

- 16 043756

Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; N1N29207R2; Н1Н29209Р2; Н1Н29214Р2; or H1H29215P2) refers to a polypeptide containing an amino acid sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) is identical or similar to the mentioned amino acid sequence, which is set forth herein ( for example, SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346, 354, 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218 or 306); if the comparison is performed using the BLAST algorithm, in which the parameters of the algorithm are chosen to obtain the largest match between corresponding sequences over the entire length of the corresponding reference sequences (e.g., expected threshold: 10; word size: 3; maximum match in query range: 0; BLOSUM matrix 62; penalties for gaps: presence 11, extension 1; conditional adjustment of the matrix of compositional contributions).

A polynucleotide variant refers to a polynucleotide comprising a nucleotide sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical to the referenced nucleotide sequence set forth herein (e.g., SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 233, 241, 249, 257, 265, 273, 281, 289, 297 , 313, 321, 329, 337, 345, 353, 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217 or 305); if the comparison is performed using the BLAST algorithm, in which the parameters of the algorithm are chosen to obtain the largest match between corresponding sequences over the entire length of the corresponding reference sequences (for example, expected threshold: 10; word size: 28; maximum match in query range: 0; pass/fail score: 1, -2; gap penalties: linear).

Anti-RGB5 antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof of the present invention, in an embodiment of the present invention comprise an immunoglobulin heavy chain variable region having at least 70% (e.g., 80%, 85%, 90%, 95 %, 99%) amino acid sequence identity with the amino acid sequence of the immunoglobulin heavy chain, the amino acid sequence of which is set forth herein, for example, in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354; and/or an immunoglobulin light chain variable region having at least 70% (e.g., 80%, 85%, 90%, 95%, 99%) amino acid sequence identity with an immunoglobulin light chain whose amino acid sequence is set forth herein, e.g. , in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, or 306.

In addition, the anti-PfRH5 antigen binding protein of the present invention may include a variant immunoglobulin polypeptide containing the amino acid sequence set forth herein except one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10) mutations, such as, for example, missense mutations (eg, conservative substitutions), nonsense mutations, deletions or insertions. For example, the present invention includes antigen binding proteins that include an immunoglobulin light chain variant containing the amino acid sequence of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218 or 306, but having one or more such mutations and/or an immunoglobulin heavy chain variant containing the amino acid sequence SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 314, 322, 330, 338, 346 or 354, but having one or more of these mutations. In an embodiment of the present invention, the anti-PfRH5 antigen binding protein comprises an immunoglobulin light chain variant comprising CDR-L1, CDR-L2, and CDR-L3, wherein one or more (e.g., 1, 2, or 3) such CDRs have one or more such mutations (e.g., conservative substitutions) compared to the sequence specifically set forth herein, and/or an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2, and CDR-H3, wherein one or more (e.g., 1 or 2 or 3) such CDRs have one or more such mutations (eg, conservative substitutions) compared to the sequence that is specifically set forth herein.

The present invention also provides anti-PfRH5 antigen binding variant proteins, for example, antibodies or antigen binding fragments thereof containing one or more CDR variants (for example, any one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3) set forth herein with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% identity or sequence similarity to them.

Embodiments of the present invention also include anti-PfRH5 antibodies and antigen binding fragments thereof that contain V _H and V _L immunoglobulin variants; or HC and LC that contain an amino acid sequence having 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, or 99%) overall amino acid sequence identity or similarity to amino acid sequences corresponding to _VH , V _L , HC or LC as specifically set forth in

- 17 043756 herein, however wherein CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of such immunoglobulins are not variants and contain the amino acid sequences set forth herein. Thus, in such embodiments, the CDRs in such antigen binding proteins are not variants themselves.

A conservatively modified variant or conservative substitution refers to a variant in which there are one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.). Such changes can often be made without significantly interfering with the biological activity of the antibody or fragment. Those skilled in the art will recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not significantly alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co. ., p. 224 ( ^4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity. The anti-RHN5 antigen binding proteins of this invention may contain immunoglobulin chains having the amino acid sequence herein, but having one or more conservatively modified variants.

Examples of amino acid groups that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acid side chains: aspartate and glutamate and 7) sulfur side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45.

In an embodiment of the present invention, the anti-PfRH5 antigen binding protein of the present invention, for example, comprising an immunoglobulin chain containing a variant amino acid sequence set forth herein, exhibits one or more of the following functional properties:

Inhibits in vitro or in vivo growth of Plasmodium falciparum in human red blood cells, e.g., as measured by parasite lactate dehydrogenase (LDH) activity (e.g., at a rate of about 51 to about 69%, or up to and including 100% (e.g., when anti-PfRH5 antigen-binding protein incubated with cells for 96 hours (compared to uninfected red blood cells), for example, when the antigen binding protein is H1H29100P, H1H29104P, H1H29127P, H1H29143P, or any combination of two such proteins (see, for example, Table 2-1).

Inhibits the in vitro or in vivo growth of Plasmodium falciparum strain 3D7 or 7G8 in human red blood cells, e.g., as measured by lactate dehydrogenase (LDH) activity of the LDH parasite, e.g., in the presence of chloroquine phosphate (CQ) (e.g., at a concentration of about 4.81 nM or 6.58 nM), for example, at a rate of from about 34 to 61% in the absence of CQ, from about 32 to 51% in the presence of 4.81 nM CQ, or from about 20 to 75% in the presence of 6.58 nM CQ.

Binds to the PfRH5 polypeptide or an antigenic fragment thereof, for example, PfRH5ANL.6his, with a KD of about 4.72 pM to about 1.67 nM at 25°C and/or from about 1.10 pM to about 1.10 nM at 37 °C, for example, as set out in table. 5-1 and 5-2 in this document.

Inhibits the in vitro or in vivo growth of Plasmodium falciparum strain D10, Dd2, 7G8, W2-mef, 3D7, HB3, FCR-1/FVO, Cam3.II or RF7 in human red blood cells, for example, as measured by parasite lactate dehydrogenase (LDH) activity ), for example, at a concentration of about 666.67 nM at the rate set out in table. 4-2, compared with uninfected red blood cells.

Blocks binding of the PfRH5 polypeptide (e.g., PfRH5ANL.6his, e.g., at a concentration of about 0.5 nM or 2.0 nM) to the basigin polypeptide (e.g., at about 1%, 10%, 20%, 30%, 40%, 50 %, 60%, 70%, 80%, 90%, 95%, 99% or 100%), e.g. PfRH5 binding to basigin that is bound to a solid matrix (e.g. ELISA plate) in which anti-PfRH5 antigen-binding the protein is present in an amount of about 100 nM. For example, basigin is represented by the amino acids Thr25-His205, for example, expressed using a C-terminal linker, DIEGRMD (SEQ ID NO: 363), followed by a portion of human IgG1 (Pro100-Lys330) and a 6X histidine tag.

Binds to the same epitope as H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; Н1Н29214Р2; or H1H29215P2, for example, binds to PfRH5, which does not have amino terminal residues M1-Y139 and contains residues K140-Q526, but does not have K247-L295 and

- 18 043756 having mutations T216A and T299A (PfRH5ANL.6his), for example, additionally including a C-terminal hexahistidine tag, for example, when measuring surface plasmon resonance.

Competes with Н1Н29089Р; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; N1N29209R2, N1N29214R2; or H1H29215P2 for binding to the PfRH5 polypeptide, for example, PfRH5ANL.6his.

The present invention includes a non-human primate (NHP) (eg, a monkey, such as an Aotus monkey) whose body contains an anti-PfRH5 antigen binding protein (eg, antibody or antigen binding fragment), such as H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; Н1Н29214Р2; or N1N29215R2. For example, a non-human primate may be introduced to, or engineered to express, an antigen-binding protein. In an embodiment of the present invention, the non-human primate is Aotus nancymaae. In an embodiment of the present invention, a non-human primate (eg, a monkey) is infected with Plasmodium falciparum (eg, strain 3D7).

The present invention includes neutralizing or antagonistic anti-PfRH5 antigen-binding proteins, eg, an antibody or antigen-binding fragment, which includes molecules that inhibit PfRH5 activity to any detectable extent. For example, neutralizing anti-PfRH5 antigen-binding protein can inhibit the growth of Plasmodium falciparum and/or block PfRH5/BSG binding.

Н1Н29089Р; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2;

Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2;

Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; Н1Н29214Р2; or H1H29215P2 refer to antigen-binding proteins, for example, antibodies and antigen-binding fragments thereof (including multispecific antigen-binding proteins) containing an immunoglobulin heavy chain variable region (VH; or a variant thereof) and an immunoglobulin light chain variable region (VL; or a variant thereof), which are presented in this document in table. 1-1; or which contain VH _; which contains their CDRs (CDR-H1 (or a variant thereof), CDR-H2 (or a variant thereof) and CDR-H3 (or a variant thereof)) and/or V _L; which contains their CDRs (CDR-L1 (or a variant thereof), CDR-L2 (or a variant thereof) and CDR-L3 (or a variant thereof)), for example, wherein the variable regions and/or CDRs of the immunoglobulin chains contain the specific amino acid sequences described below. In an embodiment of the present invention, VH is linked to an immunoglobulin heavy chain constant region (eg, IgG, such as IgG1 or IgG4) and/or VL is linked to an immunoglobulin light chain constant region (eg, kappa or lambda).

The antibodies and antigen-binding fragments of this invention contain immunoglobulin chains containing the amino acid sequences set forth herein, as well as cellular and in vitro post-translational modifications of the antibody or fragment. For example, the present invention includes antibodies and antigen-binding fragments thereof that specifically bind to PfRH5 containing the heavy and/or light chain amino acid sequences set forth herein (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3), as well as antibodies and fragments in which one or more amino acid residues are glycosylated, one or more Asn residues are deamidated, one or more residues (for example, Met, Trp and/or His) are oxidized, The N-terminal Gln is pyroglutamate (pyroE) and/or the C-terminal lysine is missing.

The present invention provides a vessel (eg, a plastic or glass vial, eg, with a cap or chromatography column, hollow needle or syringe barrel) containing an anti-PfRH5 antigen binding protein of the present invention, eg, H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; N1N29214R2 or N1N29215R2.

This invention also relates to an injection device containing one or more antigen binding proteins (eg, an antibody or antigen binding fragment) that specifically bind to PfRH5, for example, H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2;

- 19 043756

Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; Н1Н29214Р2; or H1H29215P2, or a pharmaceutical composition thereof. The injection device can be packaged in a kit. An injection device is a device that introduces a substance into the body of a subject through a parenteral route, such as intramuscularly, subcutaneously, or intravenously. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an auto-injector), which, for example, includes a barrel or cylinder for holding an injected liquid (e.g., containing an antibody or fragment thereof, or a pharmaceutical composition), a needle for piercing skin and/or blood vessels to inject fluid; and a plunger for pushing liquid out of the cylinder through the needle hole.

The present invention also relates to methods of administering an anti-PfRH5 antigen binding protein of the present invention, for example, HSh29089P; NSh29094R; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; H1H29214P2 or H1H29215P2, to a subject, comprising administering an antigen binding protein to a subject (eg, a human), for example, parenterally. For example, the method includes puncturing the subject's body with a syringe needle and introducing an antigen binding protein into the subject's body, such as a vein, artery, tumor, muscle tissue, or subcutaneous layer of the subject.

Obtaining human antibodies

Methods for producing human antibodies from transgenic mice are known in the art. Any such known methods can be used in the context of this invention to obtain human antibodies that specifically bind to PfRH5. An immunogen containing any of the following can be used to generate antibodies that specifically bind to PfRH5. In certain embodiments of this invention, antibodies of this invention are obtained from mice immunized with full-length native PfRH5, or live attenuated or inactivated virus, or DNA encoding a protein or fragment thereof. Alternatively, the PfRH5 protein or fragment thereof can be prepared using standard biochemical techniques, modified and used as an immunogen. In one embodiment of the present invention, the immunogen is a recombinantly produced PfRH5 protein or a fragment thereof. In certain embodiments of the present invention, the immunogen may be a PfRH5 polypeptide vaccine. In certain embodiments, one or more booster injections may be administered. In certain embodiments, the immunogen may be a recombinant PfRH5 polypeptide expressed in E. coli, or any other eukaryotic or mammalian cells, such as Chinese hamster ovary (CHO) cells.

Using VELOCIMMUNE® technology (see, for example, US 6596541, Regeneron Pharmaceuticals, VELOCIMMUNE®), high-affinity chimeric antibodies to PfRH5 having human variable regions and mouse constant regions can be initially isolated. VELOCIMMUNE® technology involves the creation of a transgenic mouse having a genome containing human heavy and light chain variable regions operably linked to mouse endogenous constant region loci such that the mouse produces an antibody containing the human variable region and the mouse constant region in response to antigen stimulation. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, a VELOCIMMUNE® mouse is challenged with the antigen of interest, and lymphatic cells (such as B cells) are isolated from the mice that express the antibodies. Lymph cells can be fused with a myeloma cell line to generate immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the required isotype heavy chain and light chain constant regions. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibody or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

Antibodies of interest can also be isolated from mouse B cells. Briefly, splenocytes are collected from each mouse and B cells are sorted (as described, for example, in US 2007/0280945A1) by FACS using the antigen of interest as a sorting reagent that binds and identifies reactive antibodies (antigen-positive B cells). cells). Various methods for identifying and sorting antigen-positive B cells, as well as constructing immunoglobulin gene expression cassettes by PCR to produce cells expressing recombinant antibodies, are well known in the art. See, for example, WO 20141460741, US Patent No. 7884054B2, and Liao, et al. June 2009. High-Throughput Isolation of Immunoglobulin Genes from Single Human In Cells and Expression as Monoclonal Antibodies. J Virol Methods 158(1-2): 171-9.

Initially, high-affinity chimeric antibodies were isolated, having a human variable region and a mouse constant region. Antibodies are characterized and selected for required characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the necessary human constant region to form a fully human antibody of the present invention, for example, wild type or modified IgG1 or IgG4. While the selected constant region may vary depending on the particular application, the characteristics of high affinity antigen binding and target specificity are in the variable region.

AHTu-PfRH5 antibodies containing Fc variants

In accordance with some embodiments of the present invention, anti-PfRH5 antigen-binding fragments are provided that contain an Fc domain containing one or more mutations that, for example, increase or decrease the binding of the antibody to the FcRn receptor, for example, at an acidic pH compared to a neutral pH. For example, the present invention includes anti-PfRH5 antibodies containing a mutation in the C _H 2 - or C _H 3 region of the Fc domain, where the mutation(s) increase(s) the affinity of the Fc domain for FcRn in an acidic environment (for example, in the endosome, when This pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T); or modification at position 428 and/or 433 (for example, H/L/R/S/P/Q or K) and/or 434 (for example, A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modification at position 250 and/or 428; or modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment, the modification comprises modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); 43зК (for example, Н433К) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256e); modification 250Q and 428L (for example, T250Q and M428L); and modification 307 and/or 308 (for example, 308F or 308P). In yet another embodiment, the modification comprises modification 265A (eg, D265A) and/or modification 297A (eg, N297A).

For example, the present invention includes anti-PfRH5 antigen binding proteins, for example, antibodies or antigen binding fragments comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (eg, T250Q and M248L); 252Y, 254T and 256E (for example, M252Y, S254T and T256E); 428L and 434S (for example, M428L and N434S); 257I and 311I (for example, P257I and Q311I); 257I and 434H (for example, P257I and N434H); 376V and 434H (for example, D376V and N434H); 307A, 380A and 434A (for example, T307A, E380A and N434A); and 433K and 434F (for example, Н433К and N434F).

Anti-PfRH5 antigen binding proteins, eg antibodies and antigen binding fragments thereof, which contain V _H and/or V _L as set forth herein, containing any possible combinations of the above Fc domain mutations are contemplated within the scope of this invention.

The invention also includes anti-PfRH5 antigen binding proteins, antibodies or antigen binding fragments comprising the VH set forth herein and a chimeric heavy chain constant region ( _CH ), wherein the chimeric _CH region contains segments derived from the _CH regions of more than one immunoglobulin isotype. For example, antibodies of the present invention may comprise a chimeric CH region comprising part or all of a C H 2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule in combination with part or all of a C _H 3 domain derived from a human IgG1 molecule , human IgG2 or human IgG4. In accordance with certain embodiments, the antibodies of the present invention comprise a chimeric C _H region having a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from the loop region of human IgG1, human IgG2, or human IgG4, combined with a lower loop sequence (amino acid residues at positions 228- 236 according to EU numbering) derived from the hinge region of human IgG1, human IgG2 or human IgG4. In certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. An antibody containing a chimeric C _H region as described herein can, in some embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See for example WO 2014/022540).

- 21 043756

Multispecific antigen-binding proteins

This invention includes anti-PfRH5 antigen-binding proteins, eg, antibodies and antigen-binding fragments thereof, as well as methods for using them and methods for creating such antigen-binding proteins. The term anti-PfRH5 antigen-binding proteins, e.g., antibodies or antigen-binding fragments, includes multispecific (e.g., bispecific or biparatope) molecules that contain at least one first antigen-binding domain that specifically binds PfRH5 (e.g., the antigen-binding domain from H1H29089P; N1N29094R; N1N29100R; N1N29104R; N1N29106R; N1N29109R; N1N29125R; N1N29127R; N1N29131R; N1N29134R; N1N29138R; N1N29141R; N1N29143R; N1 N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; N1N2 9196Р2;Н1Н29198Р2; H1H29207P2; H1H29209P2; H1H29214P2 or H1H29215P2) and at least one second antigen binding domain that binds to another antigen or to an epitope in PfRH5 that is different from the first antigen binding domain (for example, CD3, CD16, BSG (basigin), EXP1, MSP1, MSP2, MSPMSP3, MSP4, MSP5, MSP6, MSP7, MSP9, MSP10 GLURP, Sera, RAMA, SEA, AMA1, MTRAP, PTRAMP, ASP, RH1, RH2a, RH2b, RH4, RAP1, RAPH2, RAP3, Rhop, RhopH3, EMA175 , EMA140 and/or EBA181). In an embodiment of the present invention, the first and second epitopes overlap. In another embodiment of the present invention, the first and second epitopes do not overlap.

In an embodiment of the present invention, the multispecific antigen binding protein binds to PfRH5 and to an antigen that causes activation of immune cells, such as cytotoxic T cells, NK cells, mononuclear phagocytes or neutrophils, for example CD3 or CD16.

Н1Н29089Р; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2;

Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2;

Н1Н29198Р2; Н1Н29207Р2; Н1Н29214Р2; or H1H29215P2 includes multispecific molecules, for example antibodies or antigen binding fragments, which contain HCDR and LCDR, _VH and VL or HC and LC H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; H1H29214P2 or H1H29215P2, respectively (including variants thereof as set forth herein), and one or more antigen binding domains that bind to another epitope.

In an embodiment of the present invention, the antigen binding domain that specifically binds to PfRH5, which may be included in a multispecific molecule, comprises:

(1) (i) a heavy chain variable domain sequence that contains CDR-H1, CDRH2 and CDR-H3 from an immunoglobulin heavy chain selected from: H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; H1H29214P2 and H1H29215P2, and (ii) a light chain variable domain sequence that contains CDR-L1, CDR-L2 and CDR-L3 from an immunoglobulin heavy chain selected from: H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; Н1Н29214Р2 and Н1Н29215Р2 respectively; or (2) (i) a heavy chain variable domain (VH) sequence selected from H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; N1N29214R2 and N1N29215R2; and (ii) a light chain variable domain (VL) sequence selected from: H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; Н1Н29214Р2 and Н1Н29215Р2 respectively;

and one or more antigen-binding domains that bind to another epitope.

In an embodiment of the present invention, the multispecific antibody or fragment contains more than two different binding specificities (e.g., a trispecific molecule), for example, one or more additional antigen binding domains that are

- 22 043756 same or different from the first and/or second antigen-binding domain.

In one embodiment of the present invention, the bispecific antigen binding fragment comprises a first scFv (e.g., containing VH and V _L H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29125P; H1H29127P; H1H29131P; H 1N29134R; N1N29138R; N1N29141R; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; N1N29196R2; N1N29198R2; N 1H29207P2; H1H29209P2; H1H29214P2 and H1H29215P2) having binding specificity to the first epitope (for example, PfRH5), and a second scFv having binding specificity to a second, different epitope. For example, in an embodiment of the present invention, the first and second scFv are linked to a linker, such as a peptide linker (e.g., a GS linker such as (GGGGS) _n (SEQ ID NO: 364), where n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Other bispecific antigen binding fragments include bispecific IgG antibody F(ab) ₂ , which contains the heavy and light chain CDRs H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2;

Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2;

H1H29214P2 and H1H29215P2, and another antibody that binds to a different epitope.

Immunoconjugates

The present invention includes anti-PfRH5 antigen-binding proteins, such as antibodies or antigen-binding fragments conjugated to another fragment, for example, a therapeutic fragment (immunoconjugate) such as a toxoid (eg, diphtheria toxoid or tetanus toxoid) or an antiparasitic drug for the treatment of Plasmodium falciparum infection. In one embodiment of the present invention, the anti-PfRH5 antibody or fragment is conjugated to any of the additional therapeutic agents set forth herein. As used herein, the term immunoconjugate refers to an antigen-binding protein, such as an antibody or antigen-binding fragment, that is chemically or biologically linked to another molecule.

Treatment methods

This invention relates to methods of treating or preventing Plasmodium falciparum infection (eg, malaria) by administering a therapeutically effective amount of an anti-PfRH5 antigen-binding protein, such as an antibody or antigen-binding fragment (eg, H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H 1Н29125Р; N1N29127R; N1N29131R; N1N29134R; N1N29138R; N1N29141R; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N2916 6P2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; N1N29196R2; N1N29198R2; N1N29207R2; N1N29209R2; N1N29214R2 or N1N29215R 2) subject (for example, person) in need of such treatment or prevention. Malaria is a disease often transmitted by the bite of an infected female mosquito (such as the Anopheles mosquito), caused by infection of the host with the parasite Plasmodium falciparum. The term Plasmodium falciparum infection refers to the invasion of a subject by Plasmodium falciparum and includes malaria.

An effective or therapeutically effective dose of an anti-PfRH5 antigen binding protein, e.g., antibody or antigen binding fragment (e.g., H1H29089R; H1H29094R; H1H29100R; H1H29104R; H1H29106R; H1H29109R; H1H29125R; H1H29127R; H1H291 31Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; N1N29196R2; N1N29198R2; N 1H29207P2; H1H29209P2; H1H29214P2 or H1H29215P2), for the treatment or prevention of Plasmodium falciparum infection refers to an amount of antibody or fragment sufficient to relieve one or more several signs and/or symptoms of infection in a subject being treated, either by inducing regression or elimination of such signs and/or symptoms, or by suppressing the progression of such signs and/or symptoms. The dosage size may vary depending on the age and size of the subject to be administered, the target disease, pathological conditions, route of administration, and the like. In an embodiment of the present invention, the effective or therapeutically effective dose of an anti-PfRH5 antigen binding protein, such as an antibody or antigen binding fragment thereof, of the present invention for treating or preventing Plasmodium falciparum infection, for example, in an adult human subject is from about 1 to 150 mg/kg . Depending on the severity of the infection, the frequency and duration of treatment may be adjusted. In certain embodiments, the antigen binding protein of this invention may be administered in an initial dose followed by one or more secondary doses. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antigen binding protein in an amount that may be approximately the same or less than the amount of the initial dose, with subsequent doses separated by at least 1-3 days; no less than one week, no less than 2 weeks; not less than 3 weeks; no less than 4 weeks; no less than 5 weeks; not less than 6 weeks; no less

- 23 043756 than 7 weeks; not less than 8 weeks; not less than 9 weeks; not less than 10 weeks; not less than 12 weeks; or at least 14 weeks.

As used herein, the term subject refers to a mammal (eg, rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human, for example, in need of preventing and/or treating Plasmodium falciparum infection. The subject may have a Plasmodium falciparum infection, or be predisposed to developing a Plasmodium falciparum infection, or have an increased risk of developing such an infection. Subjects predisposed to developing Plasmodium falciparum infection, or subjects who may have an increased risk of acquiring Plasmodium falciparum infection, include subjects with a weakened immune system, for example, due to an autoimmune disease, persons receiving immunosuppressive therapy, persons suffering from human immunodeficiency syndrome ( HIV) or acquired immunodeficiency syndrome (AIDS), with certain forms of anemia that deplete or destroy white blood cells, persons receiving radiation therapy or chemotherapy, or persons suffering from an inflammatory disease. In addition, very young or elderly subjects may be predisposed. Anyone who comes into contact with or in close proximity to mosquitoes, especially in the tropics, South America, Central America, Africa, Southeast Asia and the Eastern Mediterranean region, has an increased risk of developing Plasmodium falciparum infection.

To treat or treat means administering an anti-PfRH5 antigen-binding protein, such as an antibody or antigen-binding fragment of this invention (e.g., H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29125; H1H29125; H1H291 25; N1N29131R; N1N29134R; N1N29138R; N1N29141R; N1N29143R ; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; Н1И29214Р2 or НШ29215Р2), a subject having a Plasmodium falciparum infection such that one or more signs or symptoms of infection in the subject are reduced or eliminated, for example, when Plasmodium falciparum is attenuated or substantially eliminated (eg, completely eliminated) from the subject's body.

Signs and symptoms of Plasmodium falciparum infection include anemia; bloody stool; chills, fever, sweating; to whom; convulsions; headache; jaundice; muscle pain; nausea and vomiting; enlarged spleen; jaundice; liver enlargement; rapid breathing; Plasmodium falciparum in the bloodstream, liver, or red blood cells; anemia; hemolysis; free hemoglobin in the bloodstream; hemoglobinuria; acute renal failure; acute respiratory distress syndrome (ARDS); low blood pressure; metabolic acidosis and hypoglycemia.

The present invention also includes prophylactic administration of an anti-PfRH5 antigen binding protein, such as an antibody or antigen binding fragment thereof of the present invention (e.g., H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29109125; H1H29109 R; N1N29109R; N1N29134R; N1N29138R; N1N29141R; N1N29143R ; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; N1N29196R2; N1N29198R2; N1N29207R2; N1N29209R2; N1I29214R2 or NSh29215R2) to a subject who is at risk (for example, predisposed or at increased risk ) Plasmodium falciparum infection, in order to prevent such infection. To prevent or prevent means to administer an anti-PfRH5 antigen binding protein, e.g., an antibody or antigen binding fragment of the present invention, to a subject who is not infected with Plasmodium falciparum such that the occurrence of infection in the subject is suppressed, or the likelihood or severity is reduced if the infection does occur. occurs.

Combinations and pharmaceutical compositions

This invention relates to compositions that include anti-PfRH5 antigen binding proteins and one or more ingredients; as well as methods of their use and methods of creating such compositions.

For the production of pharmaceutical compositions of anti-PfRH5 antigen-binding proteins, for example, antibodies and their antigen-binding fragments (for example, H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29125P; H1H129H129P; H1H129 127P; N1N129127; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N2N229N2; N1N2291R2; N1N29183R2; N1N229N2198; N1N29209R2; N1N29214R2 or N1N 29215P2), the antigen binding protein is mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA. (1984); Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.)

- 24 043756 (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000)

Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y. In an embodiment of the present invention, the pharmaceutical composition is sterile. Such compositions are part of this invention.

The pharmaceutical compositions of this invention contain pharmaceutically acceptable carriers, diluents, excipients and/or stabilizers, such as, for example, water, buffering agents, stabilizing agents, preservatives, isotonicizers, nonionic detergents, antioxidants and/or various other additives.

The scope of this invention includes dried, for example, lyophilized compositions containing an anti-PfRH5 antigen-binding protein, for example, an antibody or an antigen-binding fragment thereof (for example, H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29109P; H1H29 109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; N1N29134R; N1N29138R; N1N29141R; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29 179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2; Н1Н29214Р2 or НШ29215Р2), or their pharmaceutical composition, which contains a pharmaceutically acceptable carrier but essentially free of water.

In a further embodiment of the present invention, an additional therapeutic agent that is administered to a subject in combination with an anti-PfRH5 antigen binding protein, such as an antibody or an antigen binding fragment thereof (e.g., H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29125P; H 1Н29127Р; Н1Н29131Р; Н1Н29134Р ; N1N29138R; N1N29141R; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N 29183R2; N1N29187R2; N1N29192R2; N1N29196R2; N1N29198R2; N1N29207R2; N1N29209R2; N1N29214R2; or N1N29215R2), disclosed herein, is administered to the subject according to Physicians' Desk Reference 2003 (Thomson Healthcare; ^57th edition (Nov. 1, 2002)).

The route of administration of the antigen binding protein or composition thereof may vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intra-arterial.

The present invention provides methods for administering an anti-PfRH5 antigen-binding protein, for example, an antibody or an antigen-binding fragment thereof (for example, H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H2912529P; H1H2912529P; H1H 2912529R; N1N2912529R; N1N29141R; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; NSh229N2183R2; NSh229N2183R2; NSh229N2183R2; N1N29209R2; N1N292 14P2 or H1H29215P2) to a subject, comprising introducing a protein or a pharmaceutical composition or a combination thereof into the body of the subject. For example, the method includes puncturing the subject's body with a syringe needle and introducing an antigen binding protein or pharmaceutical composition, or a combination thereof, into the subject's body, such as into a vein, artery, tumor, muscle tissue, or subcutaneous layer of the subject.

The present invention provides a vessel (e.g., a plastic or glass vial, e.g., with a cap or chromatography column, hollow-hole needle, or syringe barrel) containing any of the anti-PfRH5 antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof (e.g., H1H29089P; N1N29094R; N1N29100R; N1N29104R; N1N29106R; N1N29109R; N1N29125R; N1N29127R; N1N29131R; N1N29134R; N1N29138R; N1N29141R; N1N29143R; N1 N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; N1N2 9196Р2;Н1Н29198Р2; H1H29207R2; H1H29209R2; H1H29214R2 or H1H29215R2) or a pharmaceutical composition containing a pharmaceutically acceptable carrier or a combination thereof.

This invention includes combinations comprising an anti-PfRH5 antigen-binding protein, for example, an antibody or antigen-binding fragment thereof of the invention (e.g., H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29125P; H1H29127P; H 1N29131R; N1N29134R; N1N29138R; N1N29141R; N1N29143R ; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; H1H29196R2; H1H29198R2; H1H29207R2; H1H29209R2; H1H29214R2 or H1H29215R2), in combination with one or more additional therapeutic agents. The anti-PfRH5 antigen binding protein and the additional therapeutic agent may be present in the same composition or in separate compositions. For example, in an embodiment of the present invention, the additional therapeutic agent is an antiparasitic or antimalarial therapeutic agent. In an embodiment of this invention, the additional therapeutic agent is chloroquine, atovaquone and/or proguanil, artemether and/or lumefantrine, mefloquine, quinine, quinidine, doxycycline (optionally in combination with quinine) and/or clinda

- 25 043756 mycin (optional in combination with quinine). In an embodiment of the present invention, the additional therapeutic agent is a vaccine, such as an antimalarial vaccine, for example, RTS, S/AS01 (commercially available as Mosquirix). Methods of treating or preventing Plasmodium falciparum infection in a subject in need of said treatment or prevention by administering H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2;

Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2; Н1Н29207Р2; Н1Н29209Р2;

H1H29214P2 or H1H29215P2 in combination with another therapeutic agent is part of this invention.

The present invention includes a combination comprising two or more (eg, 2, 3 or 4) antigen binding proteins of the present invention (eg, an antibody or an antigen binding protein) in combination with each other. For example, if H1H29O89P is Ab1; if H1H29094P is Ab2; if H1H29100P is Ab1; if H1H29104P is Ab4; if H1H29106P is Ab5; if H1H29109P is Ab6; if H1H29125P is Ab7; if H1H29127P is Ab8; if H1H29131P is Ab9; if H1H29134P is Ab10; if H1H29138P is Ab11; if H1H29141P is Ab12; if H1H29143P is Ab13; if NSh29146P2 is Ab14; if NSh29147P2 is Ab15; if H1H29149P2 is Ab16; if H1H29151P2 is Ab17; if HSH29163P2 is Ab18; if H1H29166P2 is Ab19; if H1H29171P2 is Ab20; if H1H29179P2 is Ab21; if NSh29183P2 is Ab22; if NSh29187P2 is Ab23; if H1H29192P2 is Ab24; if H1H29196P2 is Ab25; if H1H29198P2 is Ab26; if H1H29207P2 is Ab27; if H1H29214P2 is Ab28; if H1H29215P2 is Ab29 and if H1H29209P is Ab30, then such compositions of this invention include combinations comprising the following antigen binding proteins of this invention (e.g., antibodies and/or antigen binding proteins) in combination with each other:

- 26 043756

Abi And Abi And AB2 and ABZ And ABZ And AB4 And AB5 And ABb And AB7 and AB8 And Ab2 AB22 AI5 AB9 AB29 AB24 AB20 AI7 AI5 AI4 Abi And Abi And AB2 and ABZ And AB4 And AB4 And AB5 And ABb And AB7 and AB8 And Ab3 AB23 АИ6 AMO AB5 AB25 AB21 Аы8 АИ6 AI5 Abi And Abi And AB2 and ABZ And AB4 And AB4 And AB5 And ABb And AB7 and AB8 And Ab4 AB24 AI7 AI1 ABb AB26 AB22 Аы9 AI7 АИ6 Abi And Abi And AB2 and ABZ And AB4 And AB4 And AB5 And ABb And AB7 and AB8 And Ab5 AB25 Аы8 AI2 AB7 AB27 AB23 AB20 Аы8 AI7 Abi And Abi And AB2 and ABZ And AB4 And AB4 And AB5 And ABb And AB7 and AB8 And Ab6 AB26 Аы9 AYZ AB8 AB28 AB24 AB21 Аы9 Аы8 Abi And Abi And AB2 and ABZ And AB4 And AB4 And AB5 And ABb And AB7 and AB8 And Ab7 AB27 AB20 AI4 AB9 AB29 AB25 AB22 AB20 Аы9 Abi And Abi And AB2 and ABZ And AB4 And AB5 And AB5 And ABb And AB7 and AB8 And Ab8 AB28 AB21 AI5 AMO ABb AB26 AB23 AB21 AB20 Abi And Abi And AB2 and ABZ And AB4 And AB5 And AB5 And ABb And AB7 and AB8 And Ab9 AB29 AB22 АИ6 AI1 AB7 AB27 AB24 AB22 AB21 Abi And AB2 And AB2 and ABZ And AB4 And AB5 And AB5 And ABb And AB7 and AB8 And AblO ABZ AB23 AI7 AI2 AB8 AB28 AB25 AB23 AB22 Abi And AB2 And AB2 and ABZ And AB4 And AB5 And AB5 And ABb And AB7 and AB8 And Abll AB4 AB24 Аы8 AYZ AB9 AB29 AB26 AB24 AB23 Abi And AB2 And AB2 and ABZ And AB4 And AB5 And ABb And ABb And AB7 and AB8 And Ab 12 AB5 AB25 Аы9 AI4 AYO AB7 AB27 AB25 AB24 Abi And AB2 And AB2 and ABZ And AB4 And AB5 And ABb And ABb And AB7 and AB8 And AYZ ABb AB26 AB20 AI5 AI1 AB8 AB28 AB26 AB25 Abi And AB2 And AB2 and ABZ And AB4 And AB5 And ABb And ABb And AB7 and AB8 And Ab 14 AB7 AB27 AB21 АИ6 AI2 AB9 AB29 AB27 AB26 Abi And AB2 And AB2 and ABZ And AB4 And AB5 And ABb And AB7 And AB7 and AB8 And AI5 AB8 AB28 AB22 AI7 AYZ AYO AB8 AB28 AB27 Abi And AB2 And AB2 and ABZ And AB4 And AB5 And ABb And AB7 And AB7 and AB8 And АИ6 AB9 AB29 AB23 Аы8 AI4 AI1 AB9 AB29 AB28 Abi And AB2 And ABZ and ABZ And AB4 And AB5 And ABb And AB7 And AB8 and AB8 And AI7 AYO AB4 AB24 Аы9 AI5 AI2 AYO AB9 AB29 Abi And AB2 And ABZ and ABZ And AB4 And AB5 And ABb And AB7 And AB8 and AB9 And Аы8 Abll AB5 AB25 AB20 АИ6 AYZ AI1 AYO AYO Abi And AB2 And ABZ and ABZ And AB4 And AB5 And ABb And AB7 And AB8 and AB9 And Аы9 AI2 ABb AB26 AB21 AI7 AI4 AI2 AI1 AI1 Abi And AB2 And ABZ and ABZ And AB4 And AB5 And ABb And AB7 And AB8 and AB9 And Ab20 AYZ AB7 AB27 AB22 Аы8 AI5 AYZ AI2 AI2 Abi And AB2 And ABZ and ABZ And AB4 And AB5 And ABb And AB7 And AB8 and AB9 And Ab21 AI4 AB8 AB28 AB23 Аы9 АИ6 AI4 AYZ AYZ

- 27 043756

AB9 And Ablo and AL1 and AL2 and AYZ and AL4 and AI6 and AY8i AY9i AB22 and Ab 14 AI5 AI7 AB20 AB24 AB29 AB22 Аы9 AB29 AB25 Ab9 And Ablo and AL1 and AL2 and AYZ and AI5 and AI6 and AY8i AB20 and AB22 and AI5 АИ6 Аы8 AB21 AB25 АИ6 AB23 AB20 AB21 AB26 Ab9 And Ablo and AL1 and AL2 and AYZ and AI5 and AI6 and AI8 and AB20 and AB22 and АИ6 AI7 Аы9 AB22 AB26 AI7 AB24 AB21 AB22 AB27 Ab9 And Ablo and AL1 and AL2 and AYZ and AI5 and AI6 and AI8 and AB20 and AB22 and AI7 Аы8 AB20 AB23 AB27 Аы8 AB25 AB22 AB23 AB28 Ab9 And Ablo and AL1 and AL2 and AYZ and AI5 and AI6 and AY8i AB20 and AB22 and Аы8 Аы9 AB21 AB24 AB28 Аы9 AB26 AB23 AB24 AB29 Ab9 And Ablo and AL1 and AL2 and AYZ and AI5 and AI6 and AY8i AB20 and AB23 and Аы9 AB20 AB22 AB25 AB29 AB20 AB27 AB24 AB25 AB24 Ab9 And Ablo and AL1 and AL2 and AL4 and AI5 and AI6 and AY8i AB20 and AB23 and Ab20 AB21 AB23 AB26 AI5 AB21 AB28 AB25 AB26 AB25 Ab9 And Ablo and AL1 and AL2 and AL4 and AI5 and AYbi AY8i AB20 and AB23 and Ab21 AB22 AB24 AB27 АИ6 AB22 AB29 AB26 AB27 AB26 Ab9 And ABU and AL1 and AL2 and AL4 and AI5 and AI7 and AY8i AB20 and AB23 and Ab22 AB23 AB25 AB28 AI7 AB23 Аы8 AB27 AB28 AB27 Ab9 And ABU and AL1 and AL2 and AL4 and AI5 and AI7 and AY8i AB20 and AB23 and Ab23 AB24 AB26 AB29 Аы8 AB24 Аы9 AB28 AB29 AB28 Ab9 And AYO and AL1 and AYZ and AL4 and AI5 and AI7 and AY8i AB21 and AB23 and Ab24 AB25 AB27 AI4 Аы9 AB25 AB20 AB29 AB22 AB29 Ab9 And AYO and AL1 and AYZ and AL4 and AI5 and AI7 and AY9i AB21 and AB24 and Ab25 AB26 AB28 AI5 AB20 AB26 AB21 AB20 AB23 AB25 Ab9 And AYO and AL1 and AYZ and AL4 and AI5 and AI7 and AI9 and AB21 and AB24 and Ab26 AB27 AB29 АИ6 AB21 AB27 AB22 AB21 AB24 AB26 Ab9 And AYO and AL2 and AYZ and AL4 and AI5 and AI7 and AI9 and AB21 and AB24 and Ab27 AB28 AYZ AI7 AB22 AB28 AB23 AB22 AB25 AB27 Ab9 And AYO and AL2 and AYZ and AL4 and AI5 and AI7 and AY9i AB21 and AB24 and Ab28 AB29 AI4 Аы8 AB23 AB29 AB24 AB23 AB26 AB28 Ab9 And AL1 and AL2 and AYZ and AL4 and AI6 and AI7 and AI9 and AB21 and AB24 and Ab29 AI2 AI5 Аы9 AB24 AI7 AB25 AB24 AB27 AB29 AblO And AL1 and AL2 and AYZ and AL4 and AI6 and AI7 and AI9 and AB21 and AB25 and Abll AYZ АИ6 AB20 AB25 Аы8 AB26 AB25 AB28 AB26 Ablo and AL1 and AY2i AYZ and Аы4и AI6 and AI7 and AY9i AB21 and AB25 and Ab 12 AI4 AI7 AB21 AB26 Аы9 AB27 AB26 AB29 AB27 AblO And AL1 and AL2 and AYZ and AL4 and AI6 and AI7 and AI9 and AB22 and AB25 and AYZ AI5 Аы8 AB22 AB27 AB20 AB28 AB27 AB23 AB28 Ablo and AL1 and AL2 and AYZ and AL4 and AYbi AI7 and AY9i AB22 and AB25 and Ab 14 АИ6 Аы9 AB23 AB28 AB21 AB29 AB28 AB24 AB29

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AB26 And AI5 And AB27 ALZO AB26 And АИ6 And AB28 ALZO AB26 and AI7 And AB29 ALZO AB27 And Аы8 And AB28 ALZO AB27 And Аы9 And AB29 ALZO AB28 And AB20 And AB29 ALZO AY And AB21 And ALZO ALZO AB2 And AB22 And ALZO ALZO ABZ And AB23 And ALZO ALZO AB4 And AB24 And ALZO ALZO AB5 And AB25 And ALZO ALZO AB6 And AB26 And ALZO ALZO AB7 And AB27 And ALZO ALZO AB8 And AB28 And ALZO ALZO AB9 And AB29 And ALZO ALZO AYO And ALZO AI1 And ALZO AI2 And ALZO AYZ And ALZO AI4 And ALZO

In one embodiment of the present invention, the composition contains two or more non-competing antigen-binding proteins. Cross-competition between anti-PfRH5 antibodies of this invention is summarized below in Table. 6-1.

The term in combination with indicates that the anti-PfRH5 antigen binding protein components, such as an antibody or antigen binding fragment thereof, of the present invention, together with another agent, such as chloroquine, can be formulated into a single composition, for example, for simultaneous delivery or composed separately in two or more compositions (for example, a set including each component). Each component may be administered to a subject at a different time than when the other component is administered; for example, each administration may be performed non-simultaneously (eg, separately or sequentially) at intervals over a predetermined period of time. Moreover, the individual components may be administered to a subject by the same or a different route.

Testing and Diagnostics

Early diagnosis of malaria contributes to a positive clinical outcome. The present invention provides methods for treating a Plasmodium falciparum infection (e.g., malaria) in a subject, comprising diagnosing the infection in the subject and, if the infection is diagnosed in the subject, administering a therapeutically effective amount of an anti-PfRH5 antigen binding protein, e.g., an antibody or antigen binding fragment, to the subject . See, for example, Moody, Rapid Diagnostic Tests for Malaria Parasites, Clinical Microbiology Reviews 15 (1):66-78 (2002).

Plasmodium falciparum infection can be diagnosed under a microscope (eg, fluorescence microscopy). For example, Plasmodium falciparum can be identified by examining a drop of a subject's blood under a microscope, such as distributed as a blood smear on a microscope slide (eg, a thick blood smear or a thin blood smear). Before testing, the sample can be stained (for example, with Giemsa stain). This invention includes methods of treating

- 29 043756 Plasmodium falciparum infections (as discussed herein), in which the infection is diagnosed microscopically.

In one embodiment of the present invention, the presence of Plasmodium falciparum in a sample taken from a subject is detected by detecting Plasmodium falciparum lactate dehydrogenase. If LDH is detected higher in the test sample than in a control sample of a known uninfected sample, then the test sample is determined to contain Plasmodium falciparum. See, for example, Miura, H. Zhou, A. Diouf, SE. Moretz, M.P. Fay, L.H. Miller, L.B. Martin, M.A. Pierce, R.D. Ellis, G.E.D. Mullen, CA. Long. Anti-Apical-Membrane-Antigen-1 antibody is more effective than anti-42-kilodaltonMerozoite-Surface-Protein-1 antibody in inhibiting Plasmodium falciparum growth, as determined by the in vitro growth inhibition assay. Clin Vaccine Immunol. 16, 963-968 (2009). PMID: PMC2708396.

The presence of Plasmodium falciparum nucleic acids can also be detected, for example, by polymerase chain reaction (PCR) detecting, for example, 18S rRNA small subunit genes and/or circumsporozoites (CS).

Plasmodium falciparum infection can also be diagnosed by detecting parasite antigens in the subject's body. For example, in one embodiment of the present invention, the antigen is determined immunogenically, for example, using a rapid diagnostic test. See, for example, Van der Palen et al. Test characteristics of two rapid antigen detection tests (SD FK50 and SD FK60) for the diagnosis of malaria in returned travelers, Malaria Journal 8:90 (2009); or US Patent No. 5712170.

Anti-PfRH5 antigen-binding proteins, for example, antibodies or antigen-binding fragments thereof according to this invention (for example, H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29125P; H1H29127P; H1H29131P ; N1N29134R; N1N29138R; N1N29141R; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2 ; N1N29151R2; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; N1N29196R2; N1N29198R2; N1N29207R2; H1H29209P2; H1H29214P2 or HSh29215P2) can be used to detect and/or measure PfRH5 (for example, Plasmodium falciparum cells that contain the PfRH5 protein ) in a sample (eg, body fluid such as blood). Examples of PfRH5 assays may include, for example, contacting a sample with an anti-PfRH5 antigen binding protein of the present invention, where, for example, the anti-PfRH5 antigen binding protein is labeled with a detectable label or reporter molecule. If anti-PfRH5 antigen binding protein is detected in complex with PfRH5, this indicates the presence of PfRH5 in the sample and/or the presence of Plasmodium falciparum in the sample and in the subject.

For example, the present invention includes methods for detecting a PfRH5 polypeptide or a cell containing such a polypeptide (eg, Plasmodium falciparum) using an immunochromatographic antigen detection test. Such chromatographic tests are based on the capture of detectable labeled antigen-binding proteins (eg, antibodies and antigen-binding fragments thereof) to produce a visible band on a strip of substrate. In diagnostic tests for Plasmodium falciparum malaria, a labeled antigen binding protein first binds to the parasite antigen, PfRH5, and the resulting complex is captured on the strip by a band of bound antigen binding protein, forming a visible line (test line). The control line provides information about the integrity of the antibody-tag conjugate, but does not confirm the ability to detect parasite antigen.

The present invention provides a chromatographic test strip for detecting the presence of PfRH5 (e.g., Plasmodium falciparum cells) in a sample containing a substrate (e.g., nitrocellulose), which contains the following regions located laterally along the substrate:

(i) sample insertion site (sample area);

(ii) a conjugate spacer that contains a first anti-PfRH5 antigen-binding protein (e.g., H1H29089P; H1H29094P; H1H29100P; H1H29104P; H1H29106P; H1H29109P; H1H29125P; H1H29127P; H1H29131P; H1 N29134R; N1N29138R; N1N29141R; N1N29143R; N1N29146R2; N1N29147R2; N1N29149R2; N1N29151R2 ; N1N29163R2; N1N29166R2; N1N29171R2; N1N29179R2; N1N29183R2; N1N29187R2; N1N29192R2; N1N29196R2; N1N29198R2; N1N29207R2; N1N29209R2; H1H29214P2 or H1H29215P2), which is detectably labeled, for example, with a dye, but is not immobilized on the substrate;

(iii) a test line containing a second anti-PfRH5 antigen binding protein that is not detectably labeled and does not significantly compete with the first anti-PfRH5 antigen binding protein for binding to PfRH5, but is immobilized on a substrate; and (iv) a control line containing a secondary antigen binding protein immobilized on a substrate that binds to a first anti-PfRH5 antigen binding protein (eg, an antibody or antigen binding fragment, or protein A).

In an embodiment of this invention:

(i) after a sample containing liquid is placed on the sample pad, the sample (and any PfRH5 polypeptide it contains) diffuses by capillary action through the substrate onto the conjugation pad, then into the test line, and then into the control line ;

(ii) the PfRH5 polypeptide that reaches the conjugate spacer forms a complex with the detectable

- 30 043756 we are labeled with the first anti-PfRH5 antigen-binding protein, and the complex then diffuses into the test line, where an additional complex is formed with the second anti-PfRH5 antigen-binding protein (forming a ternary complex in which PfRH5 is bound by the specified first and second antigen-binding proteins and immobilized inside test line);

(iii) any excess first anti-PfRH5 antigen binding protein that is not immobilized on the test line continues to diffuse into the control line and binds to the secondary antigen binding protein, thereby becoming immobilized in the control line; and/or (iv) a positive test indicating the presence of PfRH5 in the sample is determined by the presence of a detectable label in the test line; a negative test result, indicating no detectable levels of PfRH5 in the sample, is determined by the absence of detectable label in the test line; and a detectable mark on the control line indicates that the test system is operating correctly.

In an embodiment of the present invention, the detectable label is a dye (e.g., indigo blue), an enzyme, ferritin, a fluorescent or colored microparticle/bead or nanoparticle/bead, or a colloidal metal (e.g., gold, selenium dye (e.g., in liposomes), or silver, for example, its colloidal particle). In an embodiment of the present invention, the detectable mark is detected visually.

In an embodiment of the present invention, the substrate is an insoluble material capable of supporting fluid flow, such as glass fiber filter paper; natural polymer materials, cellulose-based materials, filter paper, chromatography paper, nitrocellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide or cross-linked dextran.

The present invention also provides a method for determining whether a sample (eg, a biological fluid, such as, for example, blood) contains PfRH5 (eg, a cell containing PfRH5, such as Plasmodium falciparum), comprising contacting a sample area of a device based on principle of liquid spreading in a radial direction, as set forth herein, with a sample, waiting for capillary flow to carry the sample through the substrate to the control line, optionally waiting for some time, and observing the test and control lines, while the presence of a detectable mark in the test line line indicates that the sample contained PfRH5, and the presence of a detectable label in the control line indicates that the chromatography test strip is functioning correctly. The absence of a detectable label in the test line in the presence of a detectable label in the control line indicates the absence of PfRH5 in the sample. In an embodiment of the present invention, the method further comprises treating the subject from whom the sample is collected with a therapeutically effective amount of an anti-PfRH5 antigen binding protein (e.g., an antibody or antigen binding fragment) if the test indicates the presence of PfRH5 in the sample and the chromatographic test strip is functioning correctly.

Examples

The following examples are provided to provide those skilled in the art with complete disclosure and description of how to develop and apply the methods and compositions of this invention, and are not intended to limit the scope of what the inventors believe to be their invention. Efforts have been made to ensure accuracy in terms of numbers used (e.g. quantity, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25° C., and pressure is at or near atmospheric pressure.

Example 1: Preparation of human antibodies that bind to PfRH5.

Human antibodies to P. falciparum RH5 (PfRH5) were generated in VELOCIMMUNE® mice containing DNA encoding the human immunoglobulin heavy and kappa light chain variable regions. Mice were immunized with recombinant RH5 protein (P1RH5ANL). Some mice were immunized with recombinant PfRH5 protein (PfRH5®NL.6his) followed by a booster vaccine of P. falciparum merozoites isolated from strain 3D7. The antibody response was monitored using a PfRH5-specific immunoassay. If the desired immune response was achieved, splenocytes were recovered and antibodies were isolated directly from antigen-positive mouse B cells without fusion with myeloma cells, as described in US Pat. No. 2007/0280945A1, expressly incorporated herein by reference in its entirety. Using this method, several fully human anti-PfRH5 antibodies (ie, antibodies having human variable domains and human constant domains) were produced; exemplary antibodies thus prepared are designated H1H29089P; Н1Н29094Р; N1N29100R; N1N29104R; Н1Н29106Р; Н1Н29109Р; Н1Н29125Р; Н1Н29127Р; Н1Н29131Р; Н1Н29134Р; Н1Н29138Р; Н1Н29141Р; Н1Н29143Р; Н1Н29146Р2; Н1Н29147Р2; Н1Н29149Р2; Н1Н29151Р2; Н1Н29163Р2; Н1Н29166Р2; Н1Н29171Р2; Н1Н29179Р2; Н1Н29183Р2; Н1Н29187Р2; Н1Н29192Р2; Н1Н29196Р2; Н1Н29198Р2;

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Н1Н29207Р2; Н1Н29209Р2; N1N29214R2 and N1N29215R2.

The biological properties of exemplary antibodies produced in accordance with the methods of this example are described in detail in the Examples section below. The sequences of the immunoglobulin chains of antibodies are outlined below.

Table 1-1. Sequences of immunoglobulin chains according to this invention*

Antibody V CDR-H1 CDR-H2 CDR-H3 V _L CDR-L1 CDR-L2 CDR-L3 No. Name DNA PEP DNA PEP DNA PEP DNA PEP DNA PEP DNA PEP DNA PEP DNA PEP 1 Н1Н29089Р 1 2 3 4 5 6 7 8 9 10 eleven 12 13 14 15 16 2 N1N29094R 17 18 19 20 21 22 23 24 25 26 27 28 29 thirty 31 32 3 N1N29100R 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 4 N1N29104R 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 5 N1N29106R 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 6 Н1Н29109Р 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 7 Н1Н29125Р 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 8 N1N29127R 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 9 N1N29131R 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 10 N1N29134R 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 eleven Н1Н29138Р 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 12 Н1Н29141Р 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 13 Н1Н29143Р 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 14 Н1Н29146Р2 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 15 N1N29147R2 225 226 227 228 229 230 231 232 217 218 219 220 221 222 223 224 16 Н1Н29149Р2 233 234 235 236 237 238 239 240 217 218 219 220 221 222 223 224 17 N1N29151R2 241 242 243 244 245 246 247 248 217 218 219 220 221 222 223 224 18 N1N29163R2 249 250 251 252 253 254 255 256 217 218 219 220 221 222 223 224 19 N1N29166R2 257 258 259 260 261 262 263 264 217 218 219 220 221 222 223 224 20 N1N29171R2 265 266 267 268 269 270 271 272 217 218 219 220 221 222 223 224 21 Н1Н29179Р2 273 274 275 276 277 278 279 280 217 218 219 220 221 222 223 224 22 Н1Н29183Р2 281 282 283 284 285 286 287 288 217 218 219 220 221 222 223 224 23 N1N29187R2 289 290 291 292 293 294 295 296 217 218 219 220 221 222 223 224 24 Н1Н29192Р2 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 25 N1N29196R2 313 314 315 316 317 318 319 320 305 306 307 308 309 310 311 312 26 Н1Н29198Р2 321 322 323 324 325 326 327 328 305 306 307 308 309 310 311 312 27 N1N29207R2 329 330 331 332 333 334 335 336 305 306 307 308 309 310 311 312 28 N1N29214R2 337 338 339 340 341 342 343 344 305 306 307 308 309 310 311 312 29 N1N29215R2 345 346 347 348 349 350 351 352 305 306 307 308 309 310 311 312 thirty Н1Н29209Р2 353 354 355 356 357 358 359 360 305 306 307 308 309 310 311 312

The numbers corresponding to VH, CDR-H1, CDR-H2, CDR-H3, VL, CDR-L1, CDR-L2 and CDR-L3 refer to the SEQ ID NOs set forth herein. PEP refers to the amino acid sequence; DNA refers to the nucleotide sequence.

Immunoglobulin sequences and their corresponding SEQ ID NOs, which are summarized in table. 1-1 are presented below. The following sequences are also found in the Sequence Listing, which is incorporated herein by reference.

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SEQ ID NO: 1

GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCT CTGAAGATCTCCTGTAAGGGTTCTGGATA

CAGCTTTACCAGTTACTGGATCGTCTGGGTGCGCCAGATGCCCGGGAAAGGC CTGGAGTGGATGGGGATCATCTATCCTG

GTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTC AGCCGACAAGTCCATCAGCACCGCCTAC

CTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGA GACAAGATATAACTGGAACTACGGGGTT

TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 2

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIVWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAY

LQWS SLKASDTAMYYC ARQDITGTTGFD YWGQGTLVT VS S;

SEQ ID NO: 3

GGA TAC AGO TTT ACC AGT TAC TGG;

SEQ ID NO: 4

GYSFTSYW;

SEQ ID NO: 5

ATS TAT CCT GGT GAC TCT GAT ACC;

SEQ ID NO: 6

I YPGD SDT;

SEQ ID NO: 7

GCG AGA CAA GAT ATA ACT GGA ACT ACG GGG TTT GAC TAC;

SEQ ID NO: 8

ARQDITGTTGFD Y;

SEQ ID NO: 9

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCCGGGCAAGTCA

GAGCATTAGGAACTATTTGAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT AAGCTCCTGATCTATGCTGCATCCAGTT

TGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTT CACTCTCACCATCAGCAGTCTGCAACCT

GAAGATTTTGCAACTTATTTCTGTCAACAGAGTTACAGTACCCCATTCACTTT CGGCCCTGGGACCAAAGTGGATATCAA

ACGA;

- 33 043756

SEQ ID NO: 10

DIQMTQSPSSLSSASVGDRVTITCRASQSIRNYLNWYQQKPGKAPKLLIYAASSLQ SGVPSRFSGSGSGTDFTLTISSLQP

EDFATYFCQQSYSTPFTFGPGTKVDIKR;

SEQ ID NO: 11

CAG AGC ATT AGG AAC TAT;

SEQ ID NO: 12

Q SIRN Y;

SEQ ID NO: 13

GCT GCA TCC;

SEQ ID NO: 14

A AS;

SEQ ID NO: 15

CAA CAG AGT TAC AGT ACC CCA TTC ACT;

SEQ ID NO: 16

QQSYSTPFT;

SEQ ID NO: 17

CAGGTGCAGCTGGTGGAGTCTGGGGGAGACGTGGTCCAGCCTGGGAGGTCCC TGCGACTCTCCTGTTCAGGCACTGGATT

CACCTTCAGTAGCTATGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGA CTGGAATGGTGGCACTTATATCATATG

ATGGAAGTAATAAATATTATGGAGACTCCGTGAAGGGCCGATTCACCGTCTC CAGAGACAATTCCAAGAACACGCTGTCT

CTGCAAATGAACAGCCTGAAAACTGAGGACACGGCGATATATTACTGTGCGA AAGAGAGGCTTTTTGGAGTGGTCTCTTA

TTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA;

SEQ ID NO: 18

QVQLVESGGDVVQPGRSLRLSCSGTGFTFSSYAMHWVRQAPGKGLEWVALISY DGSNKYYGDSVKGRFTVSRDNSKNTLS

LQMNSLKTEDTAIYYCAKERLFGVVSYYGMDVWGQGTTVTVSS;

SEQ ID NO: 19

GGA TTC ACC TTC AGT AGC TAT GCC;

SEQ ID NO: 20

GFTFSSYA;

SEQ ID NO: 21

ATA TCA TAT GAT GGA AGT AAT AAA;

SEQ ID NO: 22

I S Y D G S Ν K;

SEQ ID NO: 23

GCG AAA GAG AGG CTT TTT GGA GTG GTC TCT TAT TAC GGT ATG GAC GTC;

- 34 043756

SEQ ID NO: 24

AKERLFGVVSYYGMDV;

SEQ ID NO: 25

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCCAGGCGAGTCA

GGACATTAATAGGGATCTAAATTGGTATCAGCAGAAATCAGGGAAAGGCCCC AAACTCCTGATCTACGATGCATCCAATT

TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAATAGATTTGGGACAGATTT TACTTTCACCATCAGCAGACTGCAGCCT

GAAGATATTGCAACATATTTCTGTCAACAGTATAAAAATCTCCCGTACACTTT TGGCCAGGGGACCAAGCTGGAGATCAA

ACGA;

SEQ ID NO: 26

DIQMTQSPSSLSSASVGDRVTITCQASQDINRDLNWYQQKSGKGPKLLIYDASNLE TGVPSRFSGNRFGTDFTFTISRLQP

EDIATYFCQQYKNLPYTFGQGTKLEIKR;

SEQ ID NO: 27

CAG GAC ATT AAT AGG GAT;

SEQ ID NO: 28

QDINRD;

SEQ ID NO: 29

GAT GCA TCC;

SEQ ID NO: 30

DAS;

SEQ ID NO: 31

CAA CAG TAT AAA AAT CTC CCG TAC ACT;

SEQ ID NO: 32

QQYKNLPYT;

SEQ ID NO: 33

CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCC TGTCCCTCACCTGCACTGTCTCTGGTGG

CTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGG AAGGGCCTGGAGTGGATTGGGATTATCT

ATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCAT TTCCGTAGACACGTCCAAGAACCAGTTC

TCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGC GAGACAGGACAGGGAGGCCCTCTTTGA

CTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 34

- 35 043756

QLQLQESGPGLVKPSETLSLTCT VSGGSIS S S S YYWGWIRQPPGKGLEWIGIIYYS GSTYYNPSLKSRVTISVDTSKNQF

SLKLS S VTAADTAVYYCARQDREALFDYWGQGTL VTVS S;

SEQ ID NO: 35

GGT GGC TCC ATC AGC AGT AGT AGT TAC TAC;

SEQ ID NO: 36

GGSISSSSYY;

SEQ ID NO: 37

ATS TAT TAT AGT GGG AGC ACC;

SEQ ID NO: 38

I Y Y S G S T;

SEQ ID NO: 39

GCG AGA CAG GAC AGG GAG GCC CTC TTT GAC TAC;

SEQ ID NO: 40

ARQDREALFDY;

SEQ ID NO: 41

GAAATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGA AAGTCACCATCACCTGCCGGGCCAGTCA

GCGCATTGGTAGTAGCTTACACTGGTACCAGCAGAAACCAGATCAGTCTCCA AAGCTCCTCATCAAGTATGCTTCCCAGT

CCTTCTCAGGGGTCCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTC ACCCTCACCATCAATAGCCTGGAAGCT

GAAGATGCTGCAACGTATTACTGTCATCAGAGTAGTACTTTACCCACCTTCGG CCAAGGGACACGACTGGAGATTAAACG

A;

SEQ ID NO: 42

EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSLHWYQQKPDQSPKLLIKYASQSFS GVPSRFSGSGSGTDFTLTINSLEA

EDAATYYCHQSSTLPTFGQGTRLEIKR;

SEQ ID NO: 43

CAG CGC ATT GGT AGT AGC;

SEQ ID NO: 44

QRIGS S;

SEQ ID NO: 45

TAT GCT TCC;

SEQ ID NO: 46

YAS;

SEQ ID NO: 47

CAT CAG AGT AGT ACT TTA CCC ACC;

SEQ ID NO: 48

- 36 043756

HQSSTLPT;

SEQ ID NO: 49

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTACAGCCTGGCAGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGATT

CAGGTTTGACGATTATGCCATGCACTGGGTCCGACAAGCTCCAGGGAAGGGC CTGGAATGGGTCTCAGGTATTAATTGGA

ATAGTGGTGGCAAAGGCTATGCGGACTCTGTGCAGGGCCGATTCACCATCTC CAGAGACAACGCCAAGAACTCCCTTTAT

CTGCAAATGAACAGTCTGAGAACTGAGGACACGGCCTTGTATTATTGTGCAA AAGATAGGGGTATAGCAGCTCGTCTTCT

CTCTCGTGATGCTTTTGATATGTGGGGCCAAGGGACAATGGTCACCGTCTCTT CA;

SEQ ID NO: 50

EVQLVESGGGLVQPGRSLRLSCAASGFRFDDYAMHWVRQAPGKGLEWVSGIN WNSGGKGYADSVQGRFTISRDNAKNSLY

LQMNSLRTEDTALYYCAKDRGIAARLLSRDAFDMWGQGTMVTVSS;

SEQ ID NO: 51

GGA TTC AGG TTT GAC GAT TAT GCC;

SEQ ID NO: 52

GFRFDDYA;

SEQ ID NO: 53

ATT AAT TGG AAT AGT GGT GGC AAA;

SEQ ID NO: 54

IN W N S G G K;

SEQ ID NO: 55

GCA AAA GAT AGG GGT ATA GCA GCT CGT CTT CTC TCT CGT GAT GCT TTT GAT ATG;

SEQ ID NO: 56

AKDRGIAARLLSRDAFDM;

SEQ ID NO: 57

GACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCTGGGGCCAGTCA

GGACGTTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAATCCCCT AAGCTCCTAATCTTTGCTGCATCCACTT

TGCAAGGTGGGATCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATT CACTCTCACAATCAGCAGCCTGCGGCCT

GAAGATTTTGCAACTTATTACTGTCAAACACCTTAATACTTACCCGTACACTTT TGGCCAGGGGACCAAGCTGGAGATCAA

ACGA;

SEQ ID NO: 58

- 37 043756

DIQLTQSPSFLSASVGDRVTTITCWASQDVSSYLAWYQQKPGKSPKLLIFAASTLQ GGIPSRFSGSGSGTEFTLTISSLRP

EDFATYYCQHLNTYPYTFGQGTKLEIKR;

SEQ ID NO: 59

CAG GAC GTE AGC AGT TAT;

SEQ ID NO: 60

Q D V S S Y;

SEQ ID NO: 61

GCT GCA TCC;

SEQ ID NO: 62

A AS;

SEQ ID NO: 63

CAA CAC CTT AAT ACT TAC CCG TAC ACT;

SEQ ID NO: 64

QHLNTYPYT;

SEQ ID NO: 65

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGTCTGGGAGGTCCC TGAGACTCTCCTGTGCAGCGTCTTCATT

CACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGTCTCCAGGCAAGGGG CTGGAGTGGGTGGCAGTTATAAGTTATG

ATGGAAGTAATAAATACTATGGAGACTTCGTGAGGGGCCGATTCACCATCTC CAGAGACAATTCCAAGAACACGCTGTAT

CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATGTATTATTGTGCGA GAGAAGTTCGTCGCTACTATTATTACGG

TATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA;

SEQ ID NO: 66

QVQLVESGGGVVQSGRSLRLSCAASSFTFSSYGMHWVRQSPGKGLEWVAVISY DGSNKYYGDFVRGRFTISRDNSKNTLY

LQMNSLRAEDTAMYYCAREVRRYYYYGMDVWGQGTTVTVSS;

SEQ ID NO: 67

TCA TTC ACC TTC AGT AGC TAT GGC;

SEQ ID NO: 68

SFTFSSYG;

SEQ ID NO: 69

ATA AGT TAT GAT GGA AGT AAT AAA;

SEQ ID NO: 70

I S Y D G S Ν K;

SEQ ID NO: 71

GCG AGA GAA GTT CGT CGC TAC TAT TAT TAC GGT ATG GAC GTC;

SEQ ID NO: 72

- 38 043756

AREVRRYYYYGMDV;

SEQ ID NO: 73

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCCAGGCGAGTCA

GGACATTAGTAATTATTTTAAATTGGTATCTGCAGAAACCAGGGAAAGCCCCT AAGCTCCTGATCTCCGATGCATCCAATT

TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTT TACTTTCACCATCAGCAGCCTGCAGCCT

GAAGATATTGCAACATATTACTGTCAACAGTATAATAATCTCCCCGCTCACTTT CGGCGGAGGGACCAAGGTGGAGATCAA

ACGA;

SEQ ID NO: 74

DIQMTQSPSSLSSASVGDRVTITCQASQDISNYLNWYLQKPGKAPKLLISDASNLET GVPSRFSGSGSGTDFTFTISSLQP

EDIATYYCQQYNNLPLTFGGGTKVEIKR;

SEQ ID NO: 75

CAG GAC ATT AGT AAT TAT;

SEQ ID NO: 76

Q D I S Ν Y;

SEQ ID NO: 77

GAT GCA TCC;

SEQ ID NO: 78

DAS;

SEQ ID NO: 79

CAA CAG TAT AAT AAT CTC CCG CTC ACT;

SEQ ID NO: 80

QQYNNLPLT;

SEQ ID NO: 81

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCC TGAGACTCTCCTGTGCAGTCTCTGGATT

CACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGC CTGGAGTGGGTCTCAGGTATTAGTTGGA

ATAGTGGTGACATAGACTATGCGGACTCTGTGAAGGGCCGATTCACCATTTC CAGAGACAACGCCAAGAACTCCCTGTAT

CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAA AAGATACCCTCTCAGGGACTGGAACTAC

GTGGTACTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 82

EVQLVESGGGLVQPGRSLRLSCAVSGFTFDDYAMHWVRQAPGKGLEWVSGISW NSGDIDYADSVKGRFTISRDNAKNSLY

- 39 043756

LQMNSLRAEDTALYYCAKDTLSGTGTTWYYFDYWGQGTLVTVSS;

SEQ ID NO: 83

GGA TTC ACC TTT GAT GAT TAT GCC;

SEQ ID NO: 84

GFTFDDYA;

SEQ ID NO: 85

ATT AGT TGG AAT AGT GGT GAC ATA;

SEQ ID NO: 86

I S W N S G D I;

SEQ ID NO: 87

GCA AAA GAT ACC CTC TCA GGG ACT GGA ACT ACG TGG TAC TAT TTT GAC TAC;

SEQ ID NO: 88

AKDTLSGTGTTWYYFDY;

SEQ ID NO: 89

GACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCTGGGGCCAGTCA

GGGTATTAGCAGTTATTTTAATCTGGTATCAGCAAAAACCAGGGAAAGCCCCT AAGCTCCTGATCTATGCTGCATCCACTT

TGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATT CACTCTCACAATCAGCAGCCTGCAGCCT

GAAGATTTTGCAACTTATTACTGTCAACAGGTGAATAGTTACCCTCTCACTTT

CGGCGGAGGGACCAAGGTGGAGATCAA

ACGA;

SEQ ID NO: 90

DIQLTQSPSFLSASVGDRVTTITCWASQGISSYLIWYQQKPGKAPKLLIYAASTLQS GVPSRFSGSGSGTEFTLTISSLQP

EDFATYYCQQVNSYPLTFGGGTKVEIKR;

SEQ ID NO: 91

CAG GGT ATT AGC AGT TAT;

SEQ ID NO: 92

QGISSY;

SEQ ID NO: 93

GCT GCA TCC;

SEQ ID NO: 94

A AS;

SEQ ID NO: 95

CAA CAG GTG AAT AGT TAC CCT CTC ACT;

SEQ ID NO: 96

QQVNSYPLT;

- 40 043756

SEQ ID NO: 97

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGATT

CACCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGA CTGGAGTGGATGGCAGTTATATCATATG

ATGGAAGTAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTC CAGAGACAATTCCAAGAACACGCTGTTT

CTGCAAATGAACAGCCTGAGACCTGAAGACACGGCTGTATATTACTGTGCGC AAGATGGCAGCTCGGCGATTTACTATTT

CTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA;

SEQ ID NO: 98

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWMAVISY DGSNKYYADSVKGRFTISRDNSKNTLF

LQMNSLRPEDTAVYYC AQDGS S AIYYF YGMD VWGQGTT VT VS S;

SEQ ID NO: 99

GGA TTC ACC TTC AGT AGT TAT GGC;

SEQ ID NO: 100

GFTFSSYG;

SEQ ID NO: 101

ATA TCA TAT GAT GGA AGT AAT AAA;

SEQ ID NO: 102

I S Y D G S Ν K;

SEQ ID NO: 103

GCG CAA GAT GGC AGC TCG GCG ATT TAC TAT TTC TAC GGT ATG GAC GTC;

SEQ ID NO: 104

AQDGSSAIYYFYGMDV;

SEQ ID NO: 105

GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTATAGGAGACA GAGTCACCATCACTTGTCGGGCGAGTCA

GGACATCAACAATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCT AAGTCCCTGATCTATGCTGCATCCAGTT

TGCAAAGTGGGGTCCCATCAAAGTTCAGCGGCAGTGGATCTGGGACAGATTT CACTCTCACCATCAGCAGCCTGCAGCCT

GAAGATTTTGCAACTTATTACTGCCTTCAGTATAATAGTTACCATCCCACTTT TGGCCAGGGGACCAAGCTGGAGATCAA

ACGA;

SEQ ID NO: 106

DIQMTQSPSSLSASIGDRVTITCRASQDINNYLAWFQQKPGKAPKSLIYAASSLQS GVPSKFSGSGSGTDFTLTISSLQP

EDFATYYCLQYNSYHPTFGQGTKLEIKR;

- 41 043756

SEQ ID NO: 107

CAG GAC ATC AAC AAT TAT;

SEQ ID NO: 108

QDINN Y;

SEQ ID NO: 109

GCT GCA TCC;

SEQ ID NO: 110

A AS;

SEQ ID NO: 111

CTT CAG TAT AAT AGT TAC CAT CCC ACT;

SEQ ID NO: 112

LQ YNS YHPT;

SEQ ID NO: 113

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCC

TGAGACTCTCCTGTGCAGCGTCTGGATT

CACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG

CTGGAGTGGGTGGCAGCTATATGGTATG

ATGGAAGTAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTC

CAGAGACAATTCCAAGAACACGCTGTAT

CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGA

GAGGGGAACATTACTATGGTTCGGGGCC

GTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 114

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAAIW

YDGSNKYYADSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYFCARGEHYYGSGPFDPWGQGTLVTVSS;

SEQ ID NO: 115

GGA TTC ACC TTC AGT AGC TAT GGC;

SEQ ID NO: 116

GFTFSSYG;

SEQ ID NO: 117

ATA TGG TAT GAT GGA AGT AAT AAA;

SEQ ID NO: 118

IW Y D G S Ν K;

SEQ ID NO: 119

GCG AGA GGG GAA CAT TAC TAT GGT TCG GGG CCG TTC GAC CCC;

SEQ ID NO: 120

ARGEHYYGSGPFDP;

SEQ ID NO: 121

- 42 043756

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCCGGGCAAGTCA

GAGCATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT AAGCTCCTGATCTTTGCTGCATCCAGTT

TACAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTT CACTCTCACCATCAGCAGTCTGCAACCT

GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTTCCCCGCTCACTTT CGGCGGAGGGACCAAGGTGGAGATCAA

ACGA;

SEQ ID NO: 122

DIQMTQSPSSLSSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIFAASSLQS GVPSRFSGSGSGTDFTLTISSLQP

EDFATYYCQQSYSSPLTFGGGTKVEIKR;

SEQ ID NO: 123

CAG AGC ATT AGC AAC TAT;

SEQ ID NO: 124

Q S I S Ν Y;

SEQ ID NO: 125

GCT GCA TCC;

SEQ ID NO: 126

A AS;

SEQ ID NO: 127

CAA CAG AGT TAC AGT TCC CCG CTC ACT;

SEQ ID NO: 128

QQSYSSPLT;

SEQ ID NO: 129

CAGGTGCAGCTGCAGGAGTCGGGGCCAGGACTGGTGAAGCCTTCACAGACCC TGTCCCTCACCTGCACTGTCTCAGGTGG

CTCCATCAGCAGTTTTGGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGG AAGGGCCTGGAGTGGATTGGGTACATCT

ATTACAGTGGGAGCATCGACTACAACCCGTCCCTCAAGAGTCGAATTACCAT ATCAGTCGACACGTCTAAGAACCAGTTC

TCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGC GAGAGAAAGGGACTACGGTGACTACTT

TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 130

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSFGYYWSWIRQHPGKGLEWIGYIYY SGSIDYNPSLKSRITISVDTSKNQF

SLKLS S VT AADT AVY YC ARERD YGD YFD YWGQGTL VT VS S;

SEQ ID NO: 131

- 43 043756

GGT GGC TCC ATC AGC AGT TTT GGT TAC TAC;

SEQ ID NO: 132

GGSISSFGYY;

SEQ ID NO: 133

ATS TAT TAC AGT GGG AGC ATS;

SEQ ID NO: 134

I Y Y S G S I;

SEQ ID NO: 135

GCG AGA GAA AGG GAC TAC GGT GAC TAC TTT GAC TAC;

SEQ ID NO: 136

ARERD YGD YFD Y;

SEQ ID NO: 137

GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTCTCTCCAGGGGAAA

GAGCCACCCTCTCCTGCAGGGCCAGTCA

GAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC

AGGCTCCTCATCTATGGTGCATCCACCA

GGGCCACTGGTGTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT

CACTCTCACCATCAGCAGTTTGCAGTCT

GAGGATTTTGCAGTTTATTCCTGTCAGCAGTATAATAACTGGCCTCTCACTTT

CGGCGGAGGGACCAAGGTGGAGATCAA

ACGA;

SEQ ID NO: 138

EIVMTQ SPATES VSPGERATLLSCRASQ S VS SNLAWYQQKPGQAPRLLIYGASTRA

TGVPARFSGSGSGTEFTLTISSLQS

EDFAVYSCQQYNNWPLTFGGGTKVEIKR;

SEQ ID NO: 139

CAG AGT GTT AGC AGC AAC;

SEQ ID NO: 140

Q S V S S N;

SEQ ID NO: 141

GGT GCA TCC;

SEQ ID NO: 142

GAS;

SEQ ID NO: 143

CAG CAG TAT AAT AAC TGG CCT CTC ACT;

SEQ ID NO: 144

QQYNNWPLT;

SEQ ID NO: 145

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCC

TGAGACTCTCCTGTGCAGCGTCTGGATT

- 44 043756

CACCTTCAGTAGCTATGGCATACACTGGGTCCGCCAGGCTCCAGGCAAGGGG CTGGAGTGGGTGGCACTTATATGGTATG

ATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTC CAGAGACAATTCCAAGAACACGCTGTAT

CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGA GAGATCAGGATTACTATGGTTCGGGGAG

TTCCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA;

SEQ ID NO: 146

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGIHWVRQAPGKGLEWVALIWY DGSNKYYADSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYC ARDQD YYGSGS S YGMD VWGQGTTVT VS S;

SEQ ID NO: 147

GGA TTC ACC TTC AGT AGC TAT GGC;

SEQ ID NO: 148

GFTFSSYG;

SEQ ID NO: 149

ATA TGG TAT GAT GGA AGT AAT AAA;

SEQ ID NO: 150

IW Y D G S Ν K;

SEQ ID NO: 151

GCG AGA GAT CAG GAT TAC TAT GGT TCG GGG AGT TCC TAC GGT ATG GAC GTC;

SEQ ID NO: 152

ARDQDYYGSGSSYGMDV;

SEQ ID NO: 153

GACATCCAGATGACCCAGTCGCCAGCCTCCCTGTCTGCATCTGTAGGAGACA GAGTCACCATCACTTGCCGGGCAAGTCA

GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT AAGCGCCTGATCTATGCTGCATCCAGTT

TGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTT CACTCTCACCATCAGCAGTCTGCAACCT

GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCTCACTTT TGGCCAGGGGACCAAGCTGGAGATCAA

ACGA;

SEQ ID NO: 154

DIQMTQSPASLSSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKRLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQP

EDFATYYCQQSYSTPLTFGQGTKLEIKR;

SEQ ID NO: 155

CAG AGC ATT AGC AGC TAT;

- 45 043756

SEQ ID NO: 156 Q S I S S Y; SEQ ID NO: 157 GCT GCA TCC; SEQ ID NO: 158 A AS; SEQ ID NO: 159 CAA CAG AGT TAC AGT ACC CCT CTC ACT; SEQ ID NO: 160 QQSYSTPLT; SEQ ID NO: 161 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCC

TGAGACTCTCCTGTGCAGCGTCTGGATT

CACCTTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG

CTGGAGTGGGTGGCAGTTATATGGTATG

ATGGAACTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTC

CAGAGACAATTCCAAGAACACGCTGTAT

CTGCAAATGATCAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGA

GAGACCCCTCAGGTGGGGACCACTACTA

TTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA; SEQ ID NO: 162 QVQLVESGGGVVQPGRSLRLSCAASGTFFSTYGMHWVRQAPGKGLEWVAVIW

YDGTNKYYADSVKGRFTISRDNSKNTLY

LQMISLRAEDTAVYYCARDPSGGDHYYYYGMDVWGQGTTVTVSS; SEQ ID NO: 163 GGA TTC ACC TTC AGT ACC TAT GGC; SEQ ID NO: 164 GFTFSTYG; SEQ ID NO: 165 ATA TGG TAT GAT GGA ACT AAT AAA; SEQ ID NO: 166 IW YDGTNK; SEQ ID NO: 167 GCG AGA GAC CCC TCA GGT GGG GAC CAC TAC TAT TAC TAC GGT ATG GAC GTC; SEQ ID NO: 168 ARDPSGGDHYYYYGMDV; SEQ ID NO: 169 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG

AATCACCATCACTTGCCAGGCGAGTCA

- 46 043756

GGACATTAGCAACTATTTAAATTGGTATCAGCAGAAGCCAGGGAAAGCCCCT AACCTCCTGATCTCCGATGCATCCGATT

TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTT TACTTTCACCATCAGCAGCCTGCAGCCT

GAAGATTTTGCAACATATTACTGTCAACAGTATGATAATATACCGATCACCTT CGGCCAAGGGACACGACTGGGAGATTAA

ACGA;

SEQ ID NO: 170

DIQMTQSPSSLSSASVGDRITITCQASQDISNYLNWYQQKPGKAPNLLISDASDLET GVPSRFSGSGSGTDFTFTISSLQP

EDFATYYCQQYDNIPITFGQGTRLEIKR;

SEQ ID NO: 171

CAG GAC ATT AGC AAC TAT;

SEQ ID NO: 172

Q D I S Ν Y;

SEQ ID NO: 173

GAT GCA TCC;

SEQ ID NO: 174

DAS;

SEQ ID NO: 175

CAA CAG TAT GAT AAT ATA CCG ATC ACC;

SEQ ID NO: 176

Q Q Y D ΝIP I T;

SEQ ID NO: 177

CAGGTGCAGCTGGTGGAGTCAGGGGGAGGCGTGGTCCAGCCTGGGAGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGATT

CACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG CTGGAGTGGGTGACATTTATATCATTTG

ATGAAAGGAATAAATACTATGCAGACTCCGTTAAGGGCCGATTCACCATCTC CAGAGACAATTCCAAGAACACGCTGTAT

CTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTATGTATTACTGTGCGA GCGAAGTCGGGTACAGTTTTGGTCATGA

TGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA;

SEQ ID NO: 178

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVTFISFD ERNKYYADSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAMYYCASEVGYSFGHDAFDIWGQGTMVTVSS;

SEQ ID NO: 179

GGA TTC ACC TTC AGT AGC TAT GGC;

SEQ ID NO: 180

- 47 043756

GFTFSSYG;

SEQ ID NO: 181

ATA TCA TTT GAT GAA AGG AAT AAA;

SEQ ID NO: 182

ISFDERNK;

SEQ ID NO: 183

GCG AGC GAA GTC GGG TAC AGT TTT GGT CAT GAT GCT TTT GAT ATC;

SEQ ID NO: 184

ASEVGYSFGHDAFDI;

SEQ ID NO: 185

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCCAGGCGAGTCA

GGACATTAGCAACTATTTAAATTGGTATCAGAAGAAACCAGGGAAAGCCCCT AAACTCCTGATCTACGATGCATCCAATT

TGGAAACAGGGGTCCCGTCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTT TACTTTCACCATCAGCAGCCTGCAGCCT

GAAGATATTGCAACATATTACTGTCAACAGTATGATAATTTCCCGCTCACTTT CGGCGGAGGGACCAAGGTGGAGATCAA

ACGA;

SEQ ID NO: 186

DIQMTQSPSSLSSASVGDRVTITCQASQDISNYLNWYQKKPGKAPKLLIYDASNLE TGVPSRFSGSGSGTDFTFTISSLQP

EDIATYYCQQYDNFPLTFGGGTKVEIKR;

SEQ ID NO: 187

CAG GAC ATT AGC AAC TAT;

SEQ ID NO: 188

QDISNY;

SEQ ID NO: 189

GAT GCA TCC;

SEQ ID NO: 190

DAS;

SEQ ID NO: 191

CAA CAG TAT GAT AAT TTC CCG CTC ACT;

SEQ ID NO: 192

QQYDNFPLT;

SEQ ID NO: 193

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGATT

CACCTTTAACAACTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGG CTGGAGTGGGTCTCAGCTATTAGTGGTA

- 48 043756

GTGGTGATAGCACATACTACTCAGACTCCGTGAAGGGCCGGTTCACCATCTC CAGAGACAATTCCAAGAACACGCTGTAT

CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGA AAGATCAGGGCCTGTATTACTATGGTTC

GGGGAGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQIDNO: 194

EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMSWVRQAPGRGLEWVSAISG SGDSTYYSDSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCAKDQGLYYYGSGSFDYWGQGTLVTVSS;

SEQIDNO: 195

GGA TTC ACC TTT AAC AAC TAT GCC;

SEQIDNO: 196

GFTFNNYA;

SEQIDNO: 197

ATT AGT GGT AGT GGT GAT AGC ACA;

SEQIDNO: 198

ISGSGDST;

SEQIDNO: 199

GCG AAA GAT CAG GGC CTG TAT TAC TAT GGT TCG GGG AGT TTT GAC TAC;

SEQIDNO: 200

AKDQGLYYYGSGSFDY;

SEQIDNO: 201

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCCGGGCAAGTCA

GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT AAGCTCCTGATCCAAGCTGCATCCAGTT

TGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATAT CACTCTCACCATCAGCAGTCTGCAACCC

GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCATTCACTTT CGGCCCTGGGACCAAAGTGGATATCAA

ACGA;

SEQIDNO: 202

DIQMTQSPSSLSSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIQAASSLQS GVPSRFSGSGSGTDITLTISSLQP

EDFATYYCQQSYSTPFTFGPGTKVDIKR;

SEQIDNO: 203

CAG AGC ATT AGC AGC TAT;

SEQIDNO: 204

Q S I S S Y;

SEQIDNO: 205

- 49 043756

GCT GCA TCC;

SEQ ID NO: 206

A AS;

SEQ ID NO: 207

CAA CAG AGT TAC AGT ACC CCA TTC ACT;

SEQ ID NO: 208

QQSYSTPFT;

SEQ ID NO: 209

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCC TGCAACTCTCCTGTGCAGCCTCTGGGTT

TGCCTTCAGCGACTCTGCTATATACTGGGTCCGCCAGGCTTCCGGGAAAGGG CTGGAGTGGGTTGGCCGCATTAGAAACA

AAGCTAATAGGTTCGCGACAGCATATGGTGCGTCGGTGAAAGGCAGGTTCAG CATACACAGAGATGATTCAAAGAACACG

GCGTATCTACAAATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACT GTGCCAGACATGGACACGATACTTTGAC

TGAGGGCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCA;

SEQ ID NO: 210

EVQLVESGGGLVQPGGSLQLSCAASGFAFSDSAIYWVRQASGKGLEWVGRIRNK ANRFATAYGASVKGRFSIHRDDSKNT

AYLQMNSLKTEDTAVYYCARHGHDTLTEGYGMDVWGQGTTVTVSS;

SEQ ID NO: 211

GGG TTT GCC TTC AGC GAC TCT GCT;

SEQ ID NO: 212

GFAFSDSA;

SEQ ID NO: 213

ATT AGA AAC AAA GCT AAT AGG TTC GCG ACA;

SEQ ID NO: 214

IRNKANRF AT;

SEQ ID NO: 215

GCC AGA CAT GGA CAC GAT ACT TTG ACT GAG GGC TAC GGT ATG GAC GTC;

SEQ ID NO: 216

ARHGHDTLTEGYGMDV;

Light chain #1 - SEQ ID NO: 217

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG AGTCACCATCACTTGCCGGGCAAGTCA

GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT AAGCTCCTGATCTATGCTGCATCCAGTT

- 50 043756

TGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTT CACTCTCACCATCAGCAGTCTGCAACCT

GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCCGATCAC CTTCGGCCAAGGGACACGACTGGAGAT

TAAA;

Light chain #1-SEQ ID NO: 218

DIQMTQSPSSLSSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQP

EDFATYYCQQSYSTPPITFGQGTRLEIK;

SEQ ID NO: 219

CAG AGC ATT AGC AGC TAT;

SEQ ID NO: 220

Q S I S S Y;

SEQ ID NO: 221

GCT GCA TCC;

SEQ ID NO: 222

A AS;

SEQ ID NO: 223

CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC;

SEQ ID NO: 224

QQSYSTPPIT;

SEQ ID NO: 225

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTAAAGAACCCTGGGTCCTCGG TGAAGGTCTCCTGCAAGGCTTCTGGAGG

CACCTTCAGCAGTTATACTATCAACTGGGTGCGACAGGCCCCTGGACAAGGG CTTGAGTGGATGGGAGGGATCATCCCTC

TCTATGGAACAGCAAACTACGCACAGAAGTTCCAGGCCAGAGTCACGATTTC CACGGACGAATCCACGAACACAGCCTAC

ATGGAACTGAGCAACCTGAGATTTGAAGACACGGCCGTGTATTTCTGTGCGA GTACACTGGAACTACGGGCTTTTGATGC

CTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA;

SEQ ID NO: 226

QVQLVQSGAEVKNPGSSVKVSCKASGGTFSSYTINWVRQAPGQGLEWMGGIIPL YGTANYAQKFQARVTISTDESTNTAY

MELSNLRFEDTAVYFCASTLELRAFDAFDIWGQGTMVTVSS;

SEQ ID NO: 227

GGA GGC ACC TTC AGC AGT TAT ACT;

SEQ ID NO: 228

GGTFSSYT;

SEQ ID NO: 229

- 51 043756

ATS ATS SST STS TAT GGA ASA GCA;

SEQIDNO: 230

P L Y G T A;

SEQIDNO: 231

GCG AGT ACA CTG GAA STA CGG GCT TTT GAT GCC TTT GAT ATC;

SEQIDNO: 232

ASTLELRAFDAFDI;

SEQIDNO: 233

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCC TGTCCCTCACCTGCACTGTCTCTGGTGG

CTCCATCAGCAGTGGTGGTTACTACTGGAACTGGATCCGCCAGCACCCAGGG AAGGGCCTGGAGTGGATTGGGTACATCT

ATTACAGTGGAAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCAT ATCAGTAGACACGTCTAAGAACCAGTTC

TCCCTGAAGCTGGGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGC GCGAGCTCCTCCTTATAACTGGTTTGA

CTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQIDNO: 234

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWNWIRQHPGKGLEWIGYIYY SGSTYYNPSLKSRVTISVDTSKNQF

SLKLGSVTAADTAVYYCARAPPYNWFDYWGQGTLVTVSS;

SEQIDNO: 235

GGT GGC TCC ATC AGC AGT GGT GGT TAC TAC;

SEQ ID NO: 236

GGSISSGGYY;

SEQIDNO: 237

ATS TAT TAC AGT GGA AGC ACC;

SEQIDNO: 238

IY Y S G S T;

SEQIDNO: 239

GCG CGA GCT CCT CCT TAT AAC TGG TTT GAC TAC;

SEQIDNO: 240

ARAPP YNWFD Y;

SEQIDNO: 241

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGATT

CACCTTCAGTGACTACTACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGG CTGGAATGGTTTCATACATTAGTAATA

GTGGTAATACCCAATACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTC CAGGGACAATGCCAAGAACTCCCTGTTT

- 52 043756

CTGCAAATGAACAGCCTGCGAGCCGAGGACACGGCCGTTTATTACTGTACGA GAGAGGGACTCGAATATAGCAGCTCGGA

GCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 242

QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMNWVRQAPGKGLEWVSYISN SGNTQYYADSVKGRFTISRDNAKNSLF

LQMNSLRAEDTAVYYCTREGLEYSSSEPFDYWGQGTLVTVSS;

SEQ ID NO: 243

GGA TTC ACC TTC AGT GAC TAC TAC;

SEQ ID NO: 244

GFTFSDYY;

SEQ ID NO: 245

ATT AGT AAT AGT GGT AAT ACC CAA;

SEQ ID NO: 246

ISSNSGNTQ;

SEQ ID NO: 247

ACG AGA GAG GGA CTC GAA TAT AGC AGC TCG GAG CCC TTT GAC TAC;

SEQ ID NO: 248

TREGLEYSSSEPFDY;

SEQ ID NO: 249

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTCAG TGAAGGTCTCCTGCAAGACTTCTGGATA

CACCTTCACCGCCTACTACATACACTGGGTGCGACAGGCCCCTGGACAAGGG CTTGAGTGGATGGGATGGATCAACCCTA

ACAATGGTGACACAAACTATGCACTGAGGTTTCAGGGCAGGGTCACCATGAC CAGGGACATGTCCATCAACACAGCCTAC

ATGGAGCTGCGCGGGGCTGAGATCTGACGACACGGCCGTGTATTATTGTGCGA GAGATGATCTAGCAGCAGCGGGTATCGG

CTGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 250

QVQLVQSGAEVKKPGASVKVSCKTSGYTFTAYYIHWVRQAPGQGLEWMGWIN PNNGDTNYALRFQGRVTMTRDMSINTAY

MELRGLRSDDTAVYYCARDDLAAAGIGWFDSWGQGTLVTVSS;

SEQ ID NO: 251

GGA TAC ACC TTC ACC GCC TAC TAC;

SEQ ID NO: 252

GYTFTAYY;

SEQ ID NO: 253

ATC AAC CCT AAC AAT GGT GAC ACA;

SEQ ID NO: 254

- 53 043756

IΝ Ρ Ν N G D Τ;

SEQ ID NO: 255

GCG AGA GAT GAT CTA GCA GCA GCG GGT ATC GGC TGG TTC GAC TCC;

SEQ ID NO: 256

ARDDLAAAGIGWFDS;

SEQ ID NO: 257

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGCTACAGCCTGGCAGGTCCC TGAGACTCTCCTGTGTAGCCTCTGGATT

CACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGC CTGGAGTGGGTCTCAGGAATTAGTTGGA

ATAGTGAAAGTATAGGCTATGCGGACTCTGTGAGGGGCCGATTCACCATTTC CAGAGACAACGCCAAGAACTCCCTGTAT

CTCCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAA AAGCCCCGTATAGTGGGACCTACTTCGA

ATACTTCCGCCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 258

EVQLVESGGGLLQPGRSLRLSCVASGFTFDDYAMHWVRQAPGKGLEWVSGISW NSESIGYADSVRGRFTISRDNAKNSLY

LQMNSLRAEDTALYYCAKAPYSGTYFEYFRHWGQGTLVTVSS;

SEQ ID NO: 259

GGA TTC ACC TTT GAT GAT TAT GCC;

SEQ ID NO: 260

GFTFDDYA;

SEQ ID NO: 261

ATT AGT TGG AAT AGT GAA AGT ATA;

SEQ ID NO: 262

I S W N S E S I;

SEQ ID NO: 263

GCA AAA GCC CCG TAT AGT GGG ACC TAC TTC GAA TAC TTC CGC CAC;

SEQ ID NO: 264

AKAPYSGTYFEYFRH;

SEQ ID NO: 265

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGATT

CACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG CTGGAGTGGGTGGCAGTTATATCATATG

ATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTC CAGAGACAATTCCAAGAACACGCTGTAT

CTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA AAGATGACTGGAACTACGAACGCCTTTGA

- 54 043756

TATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA;

SEQ ID NO: 266

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISY DGSNKYYADSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCAKDDWNYDAFDIWGQGTMVTVSS;

SEQ ID NO: 267

GGA TTC ACC TTC AGT AGC TAT GGC;

SEQ ID NO: 268

GFTFSSYG;

SEQ ID NO: 269

ATA TCA TAT GAT GGA AGT AAT AAA;

SEQ ID NO: 270

I S Y D G S N K;

SEQ ID NO: 271

GCG AAA GAT GAC TGG AAC TAC GAC GCC TTT GAT ATC;

SEQ ID NO: 272

AKDDWNYDAFDI;

SEQ ID NO: 273

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCC TGTCCCTCACCTGCACTGTCTCTGGTGG

CTCCATCAGCAGTAGTGGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGG AGGGGCCTGGAGTGGATTGGATACATCT

ATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCAT ATCAGTAGACACGTCTAAGAACCAGTTC

TCCCTGAAGCTGAACTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGC GAGAGTGGACTATGGTTCGGGGAGTTC

GTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 274

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSSGYYWSWIRQHPGRGLEWIGYIYY SGSTYYNPSLKSRVTISVDTSKNQF

SLKLNS VT AADT AVYYC ARVD YGSGS SFD YWGQGTL VT VS S;

SEQ ID NO: 275

GGT GGC TCC ATC AGC AGT AGT GGT TAC TAC;

SEQ ID NO: 276

GGSISSSGYY;

SEQ ID NO: 277

ATS TAT TAC AGT GGG AGC ACC;

SEQ ID NO: 278

IY Y S G S T;

SEQ ID NO: 279

- 55 043756

GCG AGA GTG GAC TAT GGT TCG GGG AGT TCG TTT GAC TAC;

SEQ ID NO: 280

ARVDYGSGSSFDY;

SEQ ID NO: 281

CAGGTTCAGCTGGTGCAGTCTGGACCTGAGGTGAAGAAGCCTGGGGCCTCAG TGAAGGTCTCCTGCAAGGCTTCTGGTTA

CACCTTTACCAGCTATGGCATCAGCTGGGTGCGACAGGCCCCTGGACAAGGG CTTGAGTGGCTGGGATGGATCAGCGGTT

TCAATGGTAGAACAGACTATACAGAGAAGCTCCAGGACAGAATCACCATGA CCACAGACAGATCCTCGAGCACAGCCTAC

ATGGAACTGAGGAGCCTGAGATATGACGACACGGCCGTGTATTACTGTGCGA GAGATGGACTGGAAAAACTTGGTGACTA

CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 282

QVQLVQSGPEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWLGWISG FNGRTDYTEKLQDRITMTTDRSSSTAY

MELRSLRYDDTAVYYCARDGLEKLGDYWGQGTLVTVSS;

SEQ ID NO: 283

GGT TAC ACC TTT ACC AGC TAT GGC;

SEQ ID NO: 284

GYTFTSYG;

SEQ ID NO: 285

ATC AGC GGT TTC AAT GGT AGA ACA;

SEQ ID NO: 286

ISGFNGRT;

SEQ ID NO: 287

GCG AGA GAT GGA CTG GAA AAA CTT GGT GAC TAC;

SEQ ID NO: 288

ARDGLEKLGD Y;

SEQ ID NO: 289

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGAGGTTCC TGAGACTCTCCTGTGCAGCGTCTGGATT

CACCTTCAGTAACTCTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG CTGGAGTGGGTGGCAGGAATATGGCATG

ATGGAAGTTATAAATATTATGTAGACTCCGTGAAGGGCCGATTCACCATCTC CAGAGACAATTCTAAGAACACGCTGTTT

CTGCAAATGAACAGCCTGCGAGCCGAGGACACGGCTGTATATTATTGTGCGA GAGATGATTACTATGCTTCGGGGACCAG

CGTGGACGTATGGGGCCAAGGGACCACGGTCACCGTCTCCTCA;

SEQ ID NO: 290

- 56 043756

QVQLVESGGGVVQPGRFLRLSCAASGFTFSNSGMHWVRQAPGKGLEWVAGIW HDGSYKYYVDSVKGRFTISRDNSKNTLF

LQMNSLRAEDTAVYYCARDDYYASGTSVDVWGQGTTVTVSS;

SEQ ID NO: 291

GGA TTC ACC TTC AGT AAC TCT GGC;

SEQ ID NO: 292

GFTFSNSG;

SEQ ID NO: 293

ATA TGG CAT GAT GGA AGT TAT AAA;

SEQ ID NO: 294

IW H D G S Y K;

SEQ ID NO: 295

GCG AGA GAT GAT TAC TAT GCT TCG GGG ACC AGC GTG GAC GT A;

SEQ ID NO: 296

ARDDYYASGTSVDV;

SEQ ID NO: 297

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAG TGAGGGTCTCCTGCATGGCCCTCTGGATA

CACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGG CTTGAGTGGATGGGATGGATCAACCCTA

ACAGTGGTGGCACAAAATATGCACAGAAGTTTCAGGGCAGGGTCACCATGAC CAGGGACACGTCCATCAGCACAGCCTAC

ATGGAGCTGAGCAGACTGAGATCTGACGACACGGCCGTATATTACTGTGCGA GAGAAGAAGTCGACGATTTTTGGAGTGG

TTACCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 298

QVQLVQSGAEVKKPGASVRVSCMASGYTFTGYYMHWVRQAPGQGLEWMGWI NPNSGGTKYAQKFQGRVTMTRDTSISTAY

MELSRLRSDDTAVYYCAREVDDFWSGYLDYWGQGTLVTVSS;

SEQ ID NO: 299

GGA TAC ACC TTC ACC GGC TAC TAT;

SEQ ID NO: 300

GYTFTGYY;

SEQ ID NO: 301

ATC AAC CCT AAC AGT GGT GGC ACA;

SEQ ID NO: 302

INPNSGGT;

SEQ ID NO: 303

GCG AGA GAA GAA GTC GAC GAT TTT TGG AGT GGT TAC CTT GAC TAC;

SEQ ID NO: 304

- 57 043756

AREEVDDFWSGYLDY;

Light Chain #2 - SEQ ID NO: 305

GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA GAGCCACCCTCTCCTGCAGGGCCAGTCA

GAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCT CCCAGGCTCCTCATCTATGGTGCATCCA

GCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGA CTTCACTCTCACCATCAGCAGACTGGAG

CCTGAAGATTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTTGGAC GTTCGGCCAAGGGACCAAGGTGGAAAT

CAAA;

Light Chain #2 - SEQ ID NO: 306

EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRA TGIPDRFSGSGSGTDFTLTISRLE

PEDFAVYYCQQYGSSPWTFGQGTKVEIK;

SEQ ID NO: 307

CAG AGT GTT AGC AGC AGC TAC;

SEQ ID NO: 308

Q S V S S S Y;

SEQ ID NO: 309

GGT GCA TCC;

SEQ ID NO: 310

GAS;

SEQ ID NO: 311

CAG CAG TAT GGT AGC TCA CCT TGG ACG;

SEQ ID NO: 312

QQYGSSPWT;

SEQ ID NO: 313

GAGGTGCAGCTGGTGGAGTCTGGAGGAGACTTGGTCCAGCCGGGGGGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGGTT

CGCCGTCAATGGCGACTATTTTAGTTGGGTCCGCCAGGCTCCAGGGAAGGGG CTGGAGTGGATCTCAGTTATTTATAGCA

GTGGTAACACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAG ACACAATTCCAAGAACACGCTGTATCTT

CAAATGAGCAGCCTAAGACCTGAGGACACGGCCGTGTATTACTGTGCGAGAG ACTTCCCTCCAATGTCTGGTGCGGACTA

CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 314

EVQLVESGGDL VQPGGSLRLSC AASGF AVNGD YF SWVRQ APGKGLEWIS VIYS S GNTYYADSVKGRFTISRHNSKNTLYL

- 58 043756

QMSSLRPEDTAVYYCARDFPPMSGADYWGQGTLVTVSS;

SEQ ID NO: 315

GGG TTC GCC GTC AAT GGC GAC TAT;

SEQ ID NO: 316

GF A VNGD Y;

SEQ ID NO: 317

ATT TAT AGC AGT GGT AAC ACA;

SEQ ID NO: 318

IYSSGNT;

SEQ ID NO: 319

GCG AGA GAC TTC CCT CCA ATG TCT GGT GCG GAC TAC;

SEQ ID NO: 320

ARDFPPMSGADY;

SEQ ID NO: 321

CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAG TGAAGGTCTCCTGCAAGGTTTCCGGATA

CACCCTCACTGAATTGTCCATGCACTGGGTGCGACAGGCTCCTGGAAAAGGG CTTGAATGGATGGGAGGTTTTGATCCTG

AACATGGTAAAATAATCTACGCACAGAAATTCCAGGGCAGAGTCACCATGAC CGAGGACACATCTACAGACACAGCCTAC

ATGGAACTGAGCAGCCTGAGATCTGAGGACACGGCCGTCTATTACTGTGCAA CATTTTATAACTGGAACTCCTACTACTT

CGGTATGGACGTCTGGGGCCACGGGACCACGGTCACCGTCTCCTCA;

SEQ ID NO: 322

QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFD PEHGKIIYAQKFQGRVTMTEDTSTDTAY

MELS SLRSEDTAVYYC ATFYNWNS YYFGMD VWGHGTT VT VS S;

SEQ ID NO: 323

GGA TAC ACC CTC ACT GAA TTG TCC;

SEQ ID NO: 324

GYTLTELS;

SEQ ID NO: 325

TTT GAT CCT GAA CAT GGT AAA ATA;

SEQ ID NO: 326

FDPEHGKI;

SEQ ID NO: 327

GCA ACA TTT TAT AAC TGG AAC TCC TAC TAC TTC GGT ATG GAC GTC;

SEQ ID NO: 328

ATFYNWNSYYFGMDV;

SEQ ID NO: 329

- 59 043756

GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGATT

CACCTTTAGCAGCTATGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGA CTGGAGTGGGTCTCAGCTGTTAGTGGAA

GTGCTGATATCACAAACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTC CAGAGACAATTCCAAACACACGCTGTAT

CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGA AGGATAAAGTGTATAACTGGAACTACGG

GATCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCA;

SEQ ID NO: 330

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAVSG SADITNYADSVKGRFTISRDNSKHTLY

LQMNSLRAEDT A VYYC AKDK VYNWNYGIYYGMD VWGQGTT VT VS S;

SEQ ID NO: 331

GGA TTC ACC TTT AGC AGC TAT GCC;

SEQ ID NO: 332

GFTFSSYA;

SEQ ID NO: 333

GTT AGT GGA AGT GCT GAT ATS ASA;

SEQ ID NO: 334

VSGSADIT;

SEQ ID NO: 335

GCG AAG GAT AAA GTG TAT AAC TGG AAC TAC GGG ATC TAC TAC GGT ATG GAC GTC;

SEQ ID NO: 336

AKDKVYNWNYGIYYGMDV;

SEQ ID NO: 337

CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCC TGTCCCTCACCTGCACTGTCTCTGGTGG

CTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGG AAGGGACTAGAGTGGATTGGGAGTATCT

ATTATAGTGGGAGCACCTACTACAATCCGTCCCTCAAGAGTCGAGTCACCAT ATCCGTAGACACGTCCAAGAACCAGTTC

TCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGC GAGACAAGGGAGGTGGGAGCGAGAAAA

CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 338

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYS GSTYYNPSLKSRVTISVDTSKNQF

- 60 043756

SLKLS S VTAADTAVYYCARQGRWERENFDYWGQGTLVT VS S;

SEQ ID NO: 339

GGT GGC TCC ATC AGC AGT AGT AGT TAC TAC;

SEQ ID NO: 340

GGSISSSSYY;

SEQ ID NO: 341

ATS TAT TAT AGT GGG AGC ACC;

SEQ ID NO: 342

I Y Y S G S T;

SEQ ID NO: 343

GCG AGA CAA GGG AGG TGG GAG CGA GAA AAC TTT GAC TAC;

SEQ ID NO: 344

ARQGRWERENFDY;

SEQ ID NO: 345

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTATTGAAGCCTTCGGAGACCC TGTCCCTCACCTGCGCTGTCTCTGATGA

GTCCTTCAGTGATTACTACTGGACCTGGATCCGCCAGCCCCCAGGGAAGGGG CTGGAGTGGATTGGGGAAATTACTCATA

GTGGAAGTACCCACTACAACCCGTCCCTCAAGAGCCGAGTCACCCTGTCAGT TGACACGTCCAAGAACCACTTCTCCCTG

AGCCTCAACTCTGTGACCGCCGCGGACACGGCTATTTATTACTGTGCGAGAG GCGGTGACTACGGTGGTTTACTTGACTA

CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 346

QVQLQQWGAGLLKPSETLSLTCAVSDESFSDYYWTWIRQPPGKGLEWIGEITHS GSTHYNPSLKSRVTLSVDTSKNHFSL

SLNSVTAADTAIYYCARGGDYGGLLDYWGQGTLVTVSS;

SEQ ID NO: 347

GAT GAG TCC TTC AGT GAT TAC TAC;

SEQ ID NO: 348

DESFSDYY;

SEQ ID NO: 349

ATT ACT CAT AGT GGA AGT ACC;

SEQ ID NO: 350

ITHSGST;

SEQ ID NO: 351

GCG AGA GGC GGT GAC TAC GGT GGT TTA CTT GAC TAC;

SEQ ID NO: 352

ARGGD YGGLLD Y;

SEQ ID NO: 353

- 61 043756

CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCGGAGACCC TGTCCCTCACCTGCACTGTCTCTGGTGG CTCCATCAGCAGTAGGAGTCACTACTGGGGCTGGATCCGCCAGCCCCCAGGG AAGGGGCTGGAGTGGATTGGGAGTATCT ATTATAGTGGGAGCACCTATTACAACCCGTCCCTCAAGAGTCGAGTCACCAT ATCCGTAGACACGTCCAAGAACC AGTTC TCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGC GAGACTTGGCTGGTACGCAGAGGAGGC

TTTTGAAATCTGGGGTCAAGGGACAATGGTCACCGTCTCTTCA;

SEQ ID NO: 354

QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSHYWGWIRQPPGKGLEWIGSIYYS GSTYYNPSLKSRVTISVDTSKNQF

SLKLS S VTAADTAVYYCARLGWYAEEAFEIWGQGTMVT VS S;

SEQ ID NO: 355

GGT GGC TCC ATC AGC AGT AGG AGT CAC TAC;

SEQ ID NO: 356

GGSISSRSHY;

SEQ ID NO: 357

ATS TAT TAT AGT GGG AGC ACC;

SEQ ID NO: 358 I Y Y S G S T; SEQ ID NO: 359 GCG AGA CTT GGC TGG TAC GCA GAG GAG GCT TTT GAA ATC; SEQ ID NO: 360 ARLGW Y AEE AFEI

Example 2: Growth inhibition assay to evaluate the ability of anti-PfRH5 antibodies to inhibit in vitro invasion of human erythrocytes and growth of P. falciparum parasites.

In this example, a set of four PfRH5-specific mAbs of this invention were tested alone and in combination in a standard growth inhibition assay with a single strain of Plasmodium falciparum (Dd2).

Experimental procedure

P. falciparum strain, Dd2 (BEI Resources) was first synchronized with 5% D-sorbitol according to standard protocols at a hematocrit of 3–5% and parasitemia of 1–2% 20–24 h before the start of the assay. Infected human red blood cells were obtained at an initial parasitemia of 0.4–0.7% and a hematocrit of 2%. Infected RBCs were combined with PfRH5-specific or control antibodies starting at a concentration of 666.67 nM with a serial dilution of 1:5 for each antibody or combination of antibodies. All antibodies used were human IgG1. Parasites were grown for 40–48 h until they reached the schizont stage (one complete life cycle). The growth of parasites was stopped by three washes with cold PBS. Final parasitemia was determined by measuring the lactate dehydrogenase (LDH) activity of the parasite (Miura, H. Zhou, A. Diouf, SE. Moretz, MP. Fay, LH. Miller, LB. Martin, MA. Pierce, RD. Ellis, GED. Mullen, CA. Long. Anti-Apical-Membrane Antigen-1 antibody is more effective than anti-42-kilodalton-Merozoite-Surface-Protein-1 antibody in inhibiting Plasmodium falciparum growth, as determined by the in vitro growth inhibition assay. Clin Vaccine Immunol. 16, 963-968 (2009). PMID: PMC2708396). The percentage of growth inhibition is expressed relative to uninfected red blood cells.

Generalized results and conclusions

PfRH5-specific antibodies were generated and tested in vitro in a growth inhibition assay in a laboratory-adapted strain as described above. In table Figure 2-1 shows the maximum percentage of growth inhibition for a subset of PfRH5-specific antibodies and combinations of PfRH5-specific antibodies. Individual antibodies and combinations of antibodies showed similar percentages of maximum growth inhibition, ranging from approximately 51-69%.

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Table 2-1. Generalized results of the maximum inhibitory activity of antibodies against

PfRH5 in relation to the growth of Plasmodium falciparum strain Dd2.*

Maximum growth inhibition (%)

mAb/mAb combination Dd2 H1H29127P+H1H29100P 66.33 H1H29127P+H1H29143P 51.37 H1H29127P+H1H29104P 56.96 H1H29100P+H1H29143P 68.70 H1H29100P+H1H29104P 54.04 H1H29143P+H1H29104P 58.54 H1H29100P 65.73 H1H29104P 54.12 H1H29127P 56.87 H1H29143P 56.00 human IgGl negative control antibody specific for cat allergy antigen - Fei d 1 -0.93

*Maximum growth inhibition by anti-PfRH5 antibodies compared to uninfected human erythrocytes. Growth inhibition assays were performed on erythrocytes infected with P. falciparum (mature trophozoite or early schizont) at 0.4–0.7% parasitemia. Antibodies were combined with infected red blood cells.

Parasites were grown for 40-48 hours (the time depended on the parasite strain) until they reached the schizont stage. Parasite growth was stopped by three washes with cold PBS. The lactate dehydrogenase (LDH) activity of the parasite was measured immediately after washing. The percentage of growth inhibition was expressed relative to uninfected erythrocytes. The results of one typical growth inhibition assay are shown above.

Example 3: Growth inhibition assay to evaluate the ability of anti-PfRH5 antibodies to inhibit in vitro invasion of human erythrocytes and growth of P. falciparum parasites in combination with chloroquine.

In this example, a subset of four PfRH5-specific mAbs of this invention were tested alone and in combination with chloroquine (CQ), a widely used antimalarial drug, in a standard growth inhibition assay with two laboratory strains. One strain of the Plasmodium falciparum parasite, 3D7, is sensitive to chloroquine, while strain 7G8 is resistant to the drug.

Experimental procedure

Each P. falciparum strain (BEI Resources) was first synchronized with 5% D-sorbitol according to standard protocols at 3–5% hematocrit and 1–2% parasitemia 20–24 h before the start of the assay. Infected human red blood cells were obtained at an initial parasitemia of 0.4–0.7% and a hematocrit of 2%. Infected RBCs were combined with PfRH5-specific or control antibodies starting at a concentration of 666.67 nM with a serial dilution of 1:5 for each IgG1 antibody and chloroquine at one of two concentrations of 4.91 or 6.58 nM. The two concentrations were selected based on chloroquine's IC ₂₅ , 4.91 nM, and IC ₅₀ , 6.58 nM, with the susceptible strain 3D7. Parasites were grown for 40–48 h until they reached the schizont stage (one complete life cycle). Parasite growth was stopped by three washes with cold PBS. Final parasitemia was determined by measuring the lactate dehydrogenase (LDH) activity of the parasite. The percentage of growth inhibition was expressed relative to uninfected erythrocytes.

Generalized results and conclusions

In table Figure 3-1 shows the maximum percentage of growth inhibition for each antibody alone and the antibody/chloroquine combination. Combining chloroquine with PfRH5-specific antibodies further increased the percentage of maximum growth inhibition obtained using antibodies alone in strain 3D7. Maximum growth inhibition using antibody alone was approximately 34 to 61%, while addition of 4.81 nM CQ to the mAb resulted in similar maximum growth inhibition (32 to 51%). Addition of 6.58 nM CQ to the mAb increased the range of growth inhibition from at least 20% to 59-75%. On the other hand, single antibodies and antibody/drug combinations showed similar percentages of maximum growth inhibition with strain 7G8 (mAb only: 47-51%; mAb+4.81 nM CQ: 44-53%; mAb+6.58 nM CQ : 30-52%).

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Table 3-1. Generalized results of the maximum inhibitory activity of antibodies against

PfRH5 in relation to the growth of different Plasmodium falciparum strains. *

Maximum growth inhibition (%)

Chloroquine phosphate mAb 3D7 7G8 No data Н1Н29089Р 61.06 51.10 N1N29100R 58.02 48.85 N1N29147R2 34.93 47.34 N1N29187R2 47.58 49.30 4.81 nM Н1Н29089Р 51.32 48.69 N1N29100R 46.64 53.76 N1N29147R2 31.45 44.22 N1N29187R2 44.53 48.70 6.58 nM Н1Н29089Р 75.59 52.78 N1N29100R 71.21 50.67 N1N29147R2 59.11 29.96 N1N29187R2 71.20 38.95

*Maximum growth inhibition by anti-PfRH5 antibodies compared to uninfected human erythrocytes. Growth inhibition assays were performed on erythrocytes infected with P. falciparum (mature trophozoite or early schizont) at 0.4–0.7% parasitemia. Antibodies were combined with infected red blood cells. Parasites were grown for 40-48 hours (depending on the parasite strain) until they reached the schizont stage. Parasite growth was stopped by three washes with cold PBS. Parasite lactate dehydrogenase activity was measured immediately after washing. The percentage of growth inhibition was expressed relative to uninfected erythrocytes. The results of one typical growth inhibition assay are shown above.

Example 4: Growth inhibition assay to evaluate the ability of anti-PfRH5 antibodies to inhibit in vitro invasion of human erythrocytes and growth of P. falciparum parasites.

In this example, a set of 30 PfRH5-specific mAbs of this invention were tested in a standard growth inhibition assay against a number of common laboratory strains (both sensitive and resistant to various antimalarials) and multidrug-resistant clinical lines.

Experimental procedure

Each strain of P. falciparum (BEI Resources) was first synchronized with 5% D-sorbitol according to standard protocols at a hematocrit of 3–5% and parasitemia of 1–2% 20–24 h before the start of the assay. Infected human red blood cells were obtained at an initial parasitemia of 0.4–0.7% and a hematocrit of 2%. Infected RBCs were combined with PfRH5-specific or control IgG1 antibodies starting at a concentration of 666.67 nM with a serial dilution of 1:5 for each antibody. Parasites were grown for 40–48 h until they reached the schizont stage (one complete life cycle). Parasite growth was stopped by three washes with cold PBS. Final parasitemia was determined by measuring the lactate dehydrogenase (LDH) activity of the parasite (Miura et al., Clin Vaccine Immunol. 16: 963-968 (2009). PMID: PMC2708396). The percentage of growth inhibition was expressed relative to uninfected erythrocytes.

Generalized results and conclusions

In table Figure 4-1 shows the maximum percentage of growth inhibition for each of the 30 PfRH5 specific mAbs tested at 666.67 nM. Application of several antibodies resulted in reduced growth in all laboratory-adapted and clinical strains of P. falciparum tested.

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Table 4-1. Generalized results of the maximum inhibitory activity of antibodies against

PfRH5 in relation to the growth of various Plasmodium falciparum strains.*

Maximum growth inhibition (%)

mAb D10 Dd2 7G8 W2- mef 3D7 nvz FCR- 1/FV ABOUT Cam3.I I RF7 Н1Н29089Р 82.32 71.29 70.09 66.39 67.59 59.15 65.98 56.16 28.95 N1N29094R 35.86 41.58 29.95 34.94 14.38 45.39 51.23 26.18 15.23 N1N29100R 81.73 64.60 66.40 67.29 61.80 62.61 66.89 52.44 17.02 N1N29104R 55.70 61.31 35.16 47.40 34.42 39.81 67.38 50.02 23.62 N1N29106R 74.86 53.97 55.49 48.93 57.97 48.36 54.28 51.68 27.41 Н1Н29109Р 44.79 42.05 39.04 37.92 32.22 32.24 56.74 35.91 8.24 N1N29125R 75.39 51.39 46.96 64.57 51.14 52.09 67.62 42.69 20.99 N1N29127R 74.44 64.67 61.19 53.61 63.90 59.36 61.13 47.00 25.68 Н1Н29131Р 63.00 68.75 50.30 63.16 50.61 59.45 71.77 50.18 26.66 N1N29134R 50.59 39.99 34.84 26.89 40.72 28.21 41.17 28.53 16.69 Н1Н29138Р 57.06 74.44 57.36 66.44 58.45 51.38 84.07 48.69 51.37 Н1Н29141Р 50.01 65.27 54.63 56.19 48.20 49.98 74.25 49.31 32.06 Н1Н29143Р 79.41 62.70 61.87 58.27 60.27 60.87 67.19 53.25 28.21 Н1Н29146Р2 10.78 26.90 19.07 0.57 8.57 33.87 7.32 8.18 5.97 N1N29147R2 81.44 65.31 65.20 64.71 60.27 64.85 70.32 51.93 31.44 Н1Н29149Р2 66.03 50.32 41.38 42.77 44.15 40.01 57.98 36.22 14.74 Н1Н29151Р2 38.66 57.97 47.52 53.90 35.49 31.74 53.70 26.34 18.26 N1N29163R2 71.83 61.17 62.62 63.66 55.62 56.45 64.52 46.72 10.73 N1N29166R2 70.94 60.02 54.60 61.87 53.11 54.93 69.69 55.90 19.41 N1N29171R2 10.60 7.71 17.03 0.74 4.04 0.52 11.67 5.45 12.52 Н1Н29179Р2 62.50 13.49 34.11 22.88 24.24 40.86 49.01 33.41 -2.99 Н1Н29183Р2 6.79 0.68 17.83 -0.55 5.68 4.92 16.05 8.66 -6.27 N1N29187R2 79.61 66.02 71.80 67.28 70.71 63.91 72.16 49.22 32.73 Н1Н29192Р2 8.70 15.16 12.42 4.35 -1.03 5.62 15.10 -3.38 -7.69 N1N29196R2 14.33 -1.65 23.44 17.00 6.72 26.76 21.94 6.09 3.78 N1N29198R2 4.38 10.13 5.31 -3.61 -5.40 15.53 11.08 14.66 1.53 N1N29207R2 73.90 61.30 58.68 65.21 61.84 49.30 69.23 45.30 31.06 Н1Н29209Р2 72.66 38.09 49.89 39.61 48.32 52.40 19.16 47.26 11.91 N1N29214R2 76.52 47.21 56.74 52.72 52.09 59.09 13.05 46.67 11.18 N1N29215R2 35.80 22.79 33.23 37.23 19.97 30.11 54.03 27.22 11.28

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Human IgGl negative control antibody specific for cat allergy antigen - Fei d 1 -1.42 11.02 11.68 6.66 13.18 27.50 4.04 -0.41 -6.62

^Maximum growth inhibition by anti-PfRH5 antibodies compared to uninfected human erythrocytes. Growth inhibition assays were performed on erythrocytes infected with P. falciparum (mature trophozoite or early schizont) at 0.4–0.7% parasitemia. Antibodies were combined with infected red blood cells. Parasites were grown for 40-48 hours (depending on the parasite strain) until they reached the schizont stage. Parasite growth was stopped by three washes with cold PBS. The lactate dehydrogenase (LDH) activity of the parasite was measured immediately after washing. The percentage of growth inhibition is expressed relative to uninfected red blood cells. The results of one representative growth inhibition assay for each P. falciparum strain are shown above.

Example 5. Binding kinetics according to Biacore for binding of anti-PfRH5 monoclonal antibodies to PfRH5ΔNL.his at 25 and 37°C.

In this example, the binding kinetics of various anti-PfRH5 antibodies of the present invention were determined.

Equilibrium dissociation constants (K _D ) for various PfRH5 reagents binding to purified anti-PfRH5 monoclonal antibodies were determined using a Biacore T200 real-time surface plasmon resonance biosensor. All binding assays were performed in a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v. surfactant Tween-20, pH 7.4 (HBS-ET) at 25 and 37°C. The surface of the Biacore CM4 sensor chip was first derivatized by amino immobilization with goat anti-human Fcy specific polyclonal antibody (Jackson ImmunoResearch Laboratories, cat. no. 109-005098) or rabbit anti-mouse Fc-specific polyclonal antibody (GE Healthcare catalog no. BR100838) to capture of anti-PfRH5 monoclonal IgG1 antibodies. Binding studies were performed on recombinant PfRH5 with the amino terminus M1-Y139 removed and containing residues K140Q526 but lacking K247-L295, and T216A and T299A expressed with a C-terminal hexahistidine tag (PfRH5ΔNL.6his). Various concentrations of PfRH5ΔNL.6his (3.125-50 nM; 2-fold serial dilution or 0.48-60 nM; 5-fold serial dilution) were first prepared in HBS-ET running buffer and injected over the surface with anti-human Fcy or anti-Fc mice captured anti-PfRH5 monoclonal antibody for four minutes at a flow rate of 50 μl/min, while dissociation of the monoclonal antibody-bound PfRH5 reagent was monitored for ten minutes in HBS-ET running buffer. Association rate constants ( _kα ) and dissociation rate constants (kd) were determined by fitting real-time binding sensorgrams to a mass transfer-limited 1:1 binding model using Scrubber 2.0c curve fitting software. The binding dissociation equilibrium constant (K _D ) and dissociation half-life (t1/2) were calculated from the kinetic rates as kd In (2)

K _D (Μ) = ^ka , And 1½ (min) = GO^kd

The kinetic parameters of binding of PfRH5ΔNL.6his with various anti-PfRH5 monoclonal antibodies according to this invention at 25°C and 37°C are shown in table. 5-1-5-2 respectively.

At 25°C, all anti-PfRH5 monoclonal antibodies of this invention bound to PfRH5ΔNL.6his with KD values ranging from 4.72 pM to 1.67 nM, as shown in table. 5-1. At 37°C, all anti-PfRH5 monoclonal antibodies of this invention bound to PrRH5ΔNL.6his with KD values ranging from 1.10 pM to 1.10 nM, as shown in table. 5-2.

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Table 5-1. Binding kinetic parameters for the binding of PfRH5ΔNL.6his to anti-PfRH5 monoclonal antibodies at 25 °C

Antibody mAb Capture Level (RU) 100 nM bound Ag(RU) k _a (1/Ms) Ml/s) _KD (M) C/2 (min) Н1Н29141Р 62.8±4.0 29.9 2.75E+06 1, ZOE-05 4.72E-12 888 Н1Н29209Р2 152.6±0.6 79.5 1.54E+06 1.37E-05 9.09E-12 843 N1N29125R 175.4±1.3 88.3 8D0E+05 1.00E- 05* 1.23E-11 1155 Н1Н29138Р 118.1±0.4 61.2 1.98E+07 4.04E-04 2.04E-11 28.6 N1N29106R 109.4±4.0 49.3 6.31E+06 9.93E-04 1.57E-10 I,6 N1N29134R 147.7±0.6 68.3 7.98E+05 1.00E- 05* 1.25E-11 1155 Н1Н29109Р 70.4±4.4 36.7 2D7E+06 1.01E-03 4.68E-10 I,4 N1N29100R 163.2±1.1 83.3 2.49E+06 1.96E-04 7.87E-11 59.1 N1N29127R 177.5±0.3 88.1 1.91E+06 9D2E-04 4.76E-10 12.7 Н1Н29089Р 180.4±0.9 96 1.46E+06 3.99E-04 2.73E-10 29 N1N29094R 186.1±0.9 87.2 1.07E+06 5,ZOE-04 4.98E-10 21.8 Н1Н29179Р2 171.7±1.0 66.3 1.83E+06 4.88E-03 2.66E-09 2.4 N1N29214R2 115.5±2.9 59.9 1.71E+06 2D7E-04 1.27E-10 53.3 Н1Н29131Р 103.8±5.1 52.4 ZD4E+06 2.23E-03 7D1E-10 5.2 N1N29215R2 74.5±0.4 34.6 1.56E+06 1.52E-03 9.74E-10 7.6 N1N29147R2 91.5±4.5 43.2 3.29E+06 1.67E-03 5.07E-10 6.9 N1N29163R2 93.6±0.2 38 3.02E+06 2.00E-03 6.63E-10 5.8 N1N29187R2 108.1±1.3 53.2 2D8E+06 2D9E-04 1.01E-10 52.8 Н1Н29149Р2 195.6±1.6 82.9 1.51E+06 4.4E-04 2.74E-10 27.9 N1N29207R2 204.0±2.0 104.9 1.68E+06 1.72E-03 1.03E-09 6.7 N1N29104R 139.4±2.6 58.4 1.24E+06 1, ZOE-OZ 1.05E-09 8.9 N1N29196R2 138.3±0.9 61 1.22E+06 2.21E-03 1.80E-09 5.2 Н1Н29183Р2 156.6±1.0 23 1.59E+05 1.00E- 05* 6.28E-11 1155 Н1Н29143Р 110.8±0.3 53.4 3.41E+06 2.77E-03 8DZE-10 4.2 N1N29166R2 102.5±6.6 28 4.27E+06 3.56E-03 8.34E-10 3.2 Н1Н29151Р2 122.4±0.2 26 1.48E+07 2.47E-02 1.67E-09 0.5 Н1Н29192Р2 148.3±0.6 -0.4 S.o? S.o? S.o? S.o? N1N29198R2 121.7±0.3 -0.8 S.o? S.o? S.o? S.o? Н1Н29146Р2 143.2±0.4 -1.5 S.o? S.o? S.o? S.o? N1N29171R2 144.2±0.4 0.4 S.o? S.o? S.o? S.o? Isotype control hlgGl 165.3±0.9 -1.3 S.o? S.o? S.o? S.o?

* indicates that no dissociation of PfRH5ANL.6his was observed under the current experimental conditions, and the kd value was fixed independently at 1.00E-05 when fitting the data.

$ indicates that no binding was observed under the current experimental conditions.

S.o. means there is no binding.

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Table 5-2. Binding kinetics parameters for the binding of PfRH5ANL.6his to aHmu-PfRH5 monoclonal antibodies at 37 °C

Antibody Capture level shab (RU) 100 nM bound Ag (RU) k _a (1/Ms) kd(1/s) K _D (Μ) 1½ (min) Н1Н29141Р 38.8+1.3 18.8 9.11E+06 1.00E- 05* Ι,ΙΟΕ-12 1155 Н1Н29209Р2 66.7+0.8 28.6 2.11E+06 1.00E- 05* 4.74Ε-12 1155 N1N29125R 65.8+2.6 19.9 1.58E+06 Ι,ΟΟΕ05* 6.34Ε-12 1155 Н1Н29138Р 40.3+0.2 18.7 4.26E+07 6.87Ε-04 1.61Ε-11 16.8 N1N29106R 58.6+6.5 17.3 1.60E+07 6.36Ε-04 3.97Ε-11 18.2 N1N29134R 58.9+0.6 19.8 2.71E+07 1D8E-03 4.37Ε-11 9.8 Н1Н29109Р 40.5+8.0 19.7 1.20E+07 5.51Ε-04 4.58Ε-11 21 N1N29100R 72.4+1.1 33.2 4.03E+06 2.10Ε-04 5.21Ε-11 55 N1N29127R 67.3+0.6 27.3 1.29E+07 7.02Ε-04 5.46Ε-11 16.4 Н1Н29089Р 76.0+1.9 32.7 4.04E+06 2.33Ε-04 5.77Ε-11 49.5 N1N29094R 72.1+2.1 21.2 2.05E+06 1D9E-04 5.79Ε-11 97.2 Н1Н29179Р2 79.6+1.1 15.3 2D4E+07 1.26Ε-03 5.88Ε-11 9.2 N1N29214R2 59.0+0.7 22.9 2.70E+06 1.63Ε-04 6.04Ε-11 70.9 Н1Н29131Р 64.8+0.4 26.6 1.31E+07 8.66Ε-04 6.61Ε-11 13.3

Η1Η29215Ρ2 41.9+3.3 9.9 1.40E+07 l,21E-03 8.64E-11 9.5 Η1Η29147Ρ2 54.6+1.5 24.9 l,24E+07 1D2E-03 9.01E-ll 10.3 Η1Η29163Ρ2 32.7+0.2 13 l,54E+07 l,68E-03 l,09E-10 6.9 Η1Η29187Ρ2 76.4+8.6 29 3.19E+06 6.87E-04 2D6E-10 16.8 Η1Η29149Ρ2 117.5+4.1 33.9 l,49E+06 6.47E-04 4.36E-10 17.9 Η1Η29207Ρ2 111.6+3.9 40.8 2.61E+06 l,23E-03 4.72E-10 9.4 Η1Η29104Ρ 110.3+11.5 23 l,91E+06 9.38E-04 4.92E-10 12.3 Η1Η29196Ρ2 73.6+1.6 14.1 2.50E+06 2.74E-03 l,10E-09 4.2 Η1Η29183Ρ2 78.4+1.7 3.5 Ho ^# Ho ^# Ho ^# Ho ^# Η1Η29143Ρ 41.5+0.2 12.3 Ho ^# Ho ^# Ho ^# Ho ^# Η1Η29166Ρ2 59.0+1.0 23.5 Ho ^# Ho ^# Ho ^# Ho ^# Η1Η29151Ρ2 45.0+0.2 2.7 Co ^$ Co ^$ Co ^$ Co ^$ Η1Η29192Ρ2 60.3+0.8 -1.2 Co ^$ Co ^$ Co ^$ Co ^$ Η1Η29198Ρ2 46.4+1.6 -3.2 Co ^$ Co ^$ Co ^$ Co ^$ Η1Η29146Ρ2 52.6+0.4 -2.6 Co ^$ Co ^$ Co ^$ Co ^$ Η1Η29171Ρ2 52.0+0.3 -5.6 Co ^$ Co ^$ Co ^$ Co ^$ Isotype control hlgGl 74.3+1.0 -1.2 Co ^$ Co ^$ Co ^$ Co ^$

* indicates that no dissociation of PfRH5ANL.6his was observed under the current experimental conditions, and the kd value was fixed independently at 1.00E-05 when fitting the data.

$ indicates that no binding was observed under the current experimental conditions.

* indicates that binding was observed under the current experimental conditions, but the kinetic values do not correspond to those reported in the table.

But. means mixed results.

S.o. means there is no binding.

Example 6 Octet Cross-Competition Between Different Anti-PfRH5 Monoclonal Antibodies

Binding competition between a panel of anti-PfRH5 monoclonal antibodies was determined using a real-time marker-free biolayer interferometry assay on the Octet HTX biosensor platform (Pall ForteBio Corp.).

The experiment was carried out at 25°C in a buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v. Tween-20 surfactant and 1 mg/ml BSA, pH 7.4 (HBS-EBT) with the plate shaken at 1000 rpm. To assess whether the two antibodies are able to compete with each other for binding to their respective epitopes on recombinant PfRH5 with the amino terminus M1-Y139 removed and containing residues K140-Q526 but lacking K247-L295, and T216A and T299A expressed with C- hexahistidine-terminated tag (PfRH5ANL.6his; SEQ ID: 362), approximately 1.4-2.0 nm PfRH5ANL.6his was first captured onto Octet biosensor tips coated with anti-PentaHis antibody (Fortebio Inc, no. 18-5122) by dipping biosensor tips for 60 s into wells containing 20 μg/ml PfRH5ANL.6his solution. The antigen-captured biosensor tips were then saturated with the first anti-PfRH5 monoclonal antibody (later designated mAb-1) by dipping into wells containing 50 μg/mL mAb-1 solution for 3 min. The antibodies used were IgG1. The biosensor tips were then dipped into wells containing a 50 μg/mL solution of a second anti-PfRH5 monoclonal antibody (hereafter referred to as mAb-2) for 3 min. The biosensor tips were washed in HBS-ET buffer between each stage of the experiment. The binding reaction was monitored in real time throughout the experiment, and the binding response at the end of each step was recorded. The binding reaction of mAb-2 with

PfRH5ΔNL.6h, previously complexed with mAb-1, was compared and the competitive/non-competitive behavior of various anti-PfRH5 monoclonal antibodies was determined, as shown in Table. 6-1. Table 6-1. Cross-competition between anti-PfRH5 monoclonal antibodies

mAb-Ι mAb-2, which competes with mAb-Ι N1N29127R N1N29106R N1N29134R N1N29106R N1N29127R N1N29134R N1N29134R N1N29106R N1N29127R Н1Н29143Р N1N29187R2 N1N29187R2 Н1Н29143Р Н1Н29151Р2 Н1Н29183Р2 mAb missing N1N29104R mAb missing N1N29207R2 Н1Н29109Р N1N29147R2 N1N29166R2 N1N29171R2 N1N29163R2 Н1Н29109Р N1N29207R2 N1N29147R2 N1N29166R2 N1N29171R2

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H1H29163P2 H1H29147P2 H1H29207P2 H1H29109P H1H29166P2 H1H29171P2 H1H29163P2 H1H29131P H1H29166P2 H1H29207P2 H1H29109P H1H29147P2 H1H29171P2 H1H29163P2 H1H29131P H1H29171P2 H1H29207P2 H1H29109P H1H29147P2 H1H29166P2 H1H29163P2 H1H29131P H1H29094P H1H29163P2 H1H29207P2 H1H29109P H1H29147P2 H1H29166P2 H1H29171P2 H1H29131P H1H29094P H1H29215P2 H1H29131P H1H29147P2 H1H29166P2 H1H29171P2 H1H29163P2 H1H29094P H1H29215P2 H1H29094P H1H29171P2 H1H29163P2

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H1H29131P H1H29215P2 H1H29151P2 H1H29138P H1H29215P2 H1H29163P2 H1H29131P H1H29094P H1H29151P2 H1H29125P H1H29151P2 H1H29187P2 H1H29094P H1H29215P2 H1H29198P2 H1H29125P H1H29215P2 H1H29149P2 H1H29100P H1H29209P2 H1H29179P2 H1H29214P2 H1H29089P H1H29100P H1H29149P2 H1H29209P2 H1H29179P2 H1H29214P2 H1H29089P H1H29209P2 H1H29149P2 H1H29100P H1H29179P2 H1H29214P2 H1H29089P H1H29179P2 H1H29149P2 H1H29100P H1H29209P2 H1H29214P2 H1H29089P H1H29214P2 H1H29149P2 H1H29100P H1H29209P2 H1H29179P2 H1H29089P H1H29089P H1H29149P2 H1H29100P H1H29209P2 H1H29179P2 H1H29214P2 H1H29138P H1H29094P H1H29141P mAb missing H1H29196P2 mAb missing H1H29198P2 H1H29151P2 H1H29146P2 mAb missing

Example 7 Multi-Round Growth Inhibition Assay to Evaluate Emerging Parasites Following Anti-PfRH5 Antibody Pressure

Plasmodium falciparum RH5-specific antibodies inhibit invasion of human erythrocytes over several replication cycles and do not cause mutations in the PfRH5 gene. Invasion of host erythrocytes is an important step in the life cycle of Plasmodium falciparum (P. falciparum) and malaria pathology. Multiple antimalarial drugs target asexual stages in the bloodstream, but their effectiveness is threatened by the emergence of drug-resistant strains (Arrow et al., Saving Lives, Buying Time: Economics of Malaria Drugs in the Age of Resistance. National Acamies Press (US). 254-266 (2004); Blasco et al., Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic. Nature Medicine. 23, 917-928 (2017)). In addition, antimalarial drugs have different pharmacokinetic properties. Some antimalarial drugs, such as artemisinin and quinine, are rapidly cleared from the body within one parasite's life cycle. On the other hand, hydrophobic and lipophilic antimalarial drugs are eliminated slowly, but they exhibit different rates of absorption depending on the amount of fat consumed (Arrow et al., Saving Lives, Buying Time: Economics of Malaria Drugs in an Age of

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resistance. National Academies Press (US). 254-266 (2004)).

Targeting reticulocyte binding protein homolog (RH5) with polyclonal (pAb) and monoclonal antibodies (mAb) effectively blocks parasitic invasion of several strains of P. falciparum into human erythrocytes in vitro (Wright et al., Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies. Nature. 515, 427-430 (2014); Galaway et al., PI 13 is a merozoite surface protein that binds the N-terminus of Plasmodium falciparum RH5. Nature Communications. 8, 14333 (2017)). Targeting the RH5 protein with a single antibody or a cocktail of antibodies may be necessary to create opposing selection pressures on the same target.

In addition, antibodies can compensate for the short half-life of common antimalarial drugs.

Finally, the Plasmodium parasite has developed ways to evade the host immune response that attempts to block parasite development, such as gene polymorphisms. This genetic diversity is often the result of immune pressure (Renia and Goh, Malaria Parasites: The Great Escape. Front Immunol. 7.463 (2016). PMC5098170). Whole-genome sequencing of more than 300 clinical isolates or laboratory strains of P. falciparum identified only 15 nonsynonymous PfRH5 SNPs within putative mAb epitopes, suggesting the conserved nature and importance of the protein. Immune pressure on conserved regions of the protein may limit the parasite's ability to develop escape mechanisms (Bustamante et ah, A full-length recombinant Plasmodium falciparum PfRH5 protein induces inhibitory antibodies that are effective across common PfRH5 genetic variants, Vaccine, 31, 373-9 (2013)) .

In this example, a set of four (4) RH5-specific monoclonal antibodies, each with hlgG1, of this invention were tested separately in an escape mutant assay with a single strain of Plasmodium falciparum (3D7).

Monoclonal antibodies HSh29089R, H1H291OOP, H1H29147R2, H1H29187R2, and REGN1932 were used.

Experimental procedure (including description of relevant cell lines, proteins, reagents, and device type and model): P. falciparum strain 3D7 (BEI Resources) was grown according to standard protocols at a hematocrit of 4% and parasitemia of 0.5%. Infected erythrocytes were combined with PfRH5-specific or control antibodies at a concentration corresponding to their respective IC ₅₀ value against the P. falciparum strain described above. The antibody concentration was gradually increased every 7-14 days to a final concentration corresponding to 110X their respective IC ₅₀ values. The growth medium containing the antibody was refreshed every 48 h, and fresh blood was added to the culture weekly.

Every week, parasite RNA was extracted by lysing infected erythrocytes with Trizol and purified using a Qiagen RNeasy kit. Reverse transcription was completed using a high-throughput cDNA reverse transcription kit (Applied Biosystems). Amplification of the RH5 gene was performed using PfRH5-specific primers. PCR products were analyzed on a 1.5% agarose gel and cloned into the TOPO TA cloning vector (Life Technologies). Sequencing of RH5 was performed using forward and reverse primers for M13 sequencing.

Summary of Results and Conclusions Several groups have reported that targeting reticulocyte binding protein homologue 5 (PfRH5) can effectively block parasitic invasion of human erythrocytes in vitro by P. falciparum. A gradual increase in the pressure of PfRH5-specific antibodies against 3D7 P. falciparum parasites did not lead to PfRH5 polymorphisms compared with the pressure of the isotype control antibody. In table Figure 7-1 shows the percentage of PfRH5 sequence identity scores of each sample compared to all other sequenced samples after 45 days of gradual increase in antibody pressure (from 1 x EC ₅₀ to 110 x EC ₅₀ ). All sequences were 100% identical across all sites. In fig. 1 depicts an alignment of PfRH5 sequences corresponding to each PfRH5-specific antibody 45 days after a gradual increase in antibody pressure (from 1 x EC ₅₀ to 110 x EC ₅₀ ), showing no sequence differences at the nucleotide level.

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Table 7-1. Percentage of PfRH5 sequence identity scores of each sample compared to all other sequenced samples after 45 days of antibody pressure ramping (1 x _EU50 to 110 x _EU50 )

Example 8: Growth Inhibition Assay to Evaluate the Ability of Anti-PfRH5 Antibodies to Inhibit In Vitro Invasion of Human Red Blood Cells and Growth of P. falciparum Parasites in the Presence of Blood Serum

Specific antibodies to Plasmodium falciparum RH5 inhibit human erythrocyte invasion in a pLDH-based growth inhibition assay in the presence of serum.

One group has proposed that activation of complement on the surface of merozoites enhances the parasite's ability to invade red blood cells (Biryukov et al., Complement and Antibody-mediated Enhancement of Red Blood Cell Invasion and Growth of Malaria Parasites. EBioMedicine. 9, 207-216 (2016)) . However, other studies indicate that the presence of serum with active complement results in reduced or comparable parasite growth compared to serum with inactive complement (Boyle et al, Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria. Immunity. 42, 580-90 (2015); Chulay et al., Inhibition of in vitro growth of Plasmodium falciparum by immune serum from monkeys. J Infect Dis. 144, 270-278). In addition, in all cases of human vaccination with merozoite antigens (or any malarial antigens), there are no documented cases of antibody-dependent increases in parasitemia.

In this example, a set of four (4) RH5-specific mAbs (each with hlgG1 (prefixed H1H) or hIgG4 (prefixed H4H)) of the invention were tested alone and in combination with heat-inactivated normal Aotus monkey serum (ANS). Aotus monkey blood serum (AHIS), human normal serum (HNS), or heat-inactivated human serum (HHIS) in a standard Plasmodium falciparum single-strain growth inhibition assay (FCR-1/FVO).

The monoclonal antibodies used were as follows: H1H29O89P, H1H29100P,

H1H29147R2, H1H29187R2, H4H29089R, H4H29100R, H4H29147R2, H4H29187R2, REGN1932 (anti-Fel d1 (human IgG1)) and REGN1945 (anti-Fel d1 (human IgG4)).

Experimental procedure (including description of relevant cell lines, proteins, reagents, and device type and model). P. falciparum strain FCR-1/FVO (BEI Resources) was first synchronized with 5% D-sorbitol according to standard protocols at a hematocrit of 35% and parasitemia of 1–2% 20–24 h before the start of the assay. Infected human red blood cells were obtained at an initial parasitemia of 0.4–0.7% and a hematocrit of 2%. Infected erythrocytes were combined with RH5-specific or control antibodies at a concentration of 6.67 μM in the presence of 10% normal Aotus blood serum, heat-inactivated Aotus blood serum, normal human blood serum, or heat-inactivated blood serum. Parasites were grown for 40–48 h until they reached the schizont stage (one complete life cycle). Parasite growth was stopped by three washes with cold PBS. Terminal parasitemia was determined by measuring the lactate dehydrogenase (LDH) activity of the parasite (Miura et al., Anti-Apical-Membrane-Antigen-1 antibody is more effective than anti-42-kilodalton-Merozoite-Surface-Protein-1 antibody in inhibiting Plasmodium falciparum growth , as determined by the in vitro growth inhibition assay. Clin Vaccine Immunol. 16, 963-968 (2009). The percentage of growth inhibition is expressed relative to uninfected red blood cells.

Generalized results and conclusions

Targeting reticulocyte binding protein homolog 5 (RH5) effectively blocks parasitic invasion of human erythrocytes in vitro by P. falciparum. Conflicting data have been published regarding the role of complement in merozoite invasion of erythrocytes. RH5-specific antibodies were generated and tested in the presence of Aotus or human serum in vitro in a growth inhibition assay in a P. falciparum strain as described above. In table 8-1 shows the percentage of maximum suppression

- 73 043756 growth for each RH5-specific antibody (both hlgG1 and hIgG4 formats) with active or inactive serum complement. Individual antibodies and combinations of active or inactive serum showed similar percentages of maximum growth inhibition, ranging from approximately 67-86%.

Table 8-1. Summarized results of maximum growth inhibitory activity of PfRH5 antibodies in the presence of 10% Aotus or human serum

Percentage of maximum growth inhibition

mAb mAb (6.67 µM) + ANS (10%) mAb (6.67 µM) + AHIS (10%) mAb (6.67 µM) + HNS (10%) mAb (6.67 µM) + HHIS (10%) mAb (6.67 mkM) + without serum and blood Н1Н29089Р 75.7 84.3 85.0 85.2 85.3 N1N29100R 73.2 82.5 82.4 81.9 83.9 Н1Н29147Р 2 80.3 86.2 85.6 83.4 84.1 N1N29187R 2 80.1 89.4 85.9 85.4 85.3 N4N29089R 75.7 82.4 85.3 86.2 84.8 N4N29100R 67.7 79.8 75.5 79.6 83.9 N4N29147R 2 67.0 76.8 73.0 75.5 82.4 N4N29187R 2 69.9 82.9 77.0 77.8 84.0 REGN1932 0 0 0 0 0 REGN1945 0 0 0 0 0

Maximum inhibition of anti-PfRH5 antibody growth compared to uninfected human erythrocytes. Growth inhibition assays were performed on erythrocytes infected with P. falciparum (mature trophozoite or early schizont) at 0.4–0.7% parasitemia. Antibodies and sera were combined with infected red blood cells. Parasites were grown for 40-48 hours (the time depended on the parasite strain) until they reached the schizont stage. Parasite growth was stopped by three washes with cold PBS. The lactate dehydrogenase (LDH) activity of the parasite was measured immediately after washing. The percentage of growth inhibition is expressed relative to uninfected red blood cells. The results of one typical growth inhibition assay are shown above. ANS: Aotus Monkey Normal Serum, AHIS: Aotus Heat Inactivated Blood Serum, HNS: Human Normal Serum, HHIS: Heat Inactivated Human Blood Serum.

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All literature cited herein is incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent had been expressly and separately identified for inclusion by reference . This incorporation by reference statement is intended by applicants to address each individual publication, database entry (e.g., Genbank sequences or GeneID records), patent application, or patent, even if such reference is not directly adjacent to the specific incorporation statement via link. The inclusion in the description of specific incorporation by reference statements, if any, does not in any way weaken the said general incorporation by reference statement. Citation of literature in this document is not intended as an admission that the literature is prior art, nor is it an admission of the content or date of those publications or documents.

Claims (28)

1. An antibody or antigen binding fragment thereof that specifically binds to a Plasmodium falciparum reticolocyte binding protein homolog 5 (PfRH5) polypeptide, wherein the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 containing the amino acid sequences of SEQ ID NO: 4 , 6 and 8, respectively, and three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, containing the amino acid sequences SEQ ID NO: 12, 14 and 16, respectively. 2. The antibody or antigen-binding fragment according to claim 1, where the antibody or antigen-binding fragment contains a heavy chain variable region (HCVR) containing an amino acid sequence - 74 043756 sequence SEQ ID NO: 2. 3. The antibody or antigen binding fragment according to claim 1, wherein the antibody or antigen binding fragment comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10. 4. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2, and an LCVR containing the amino acid sequence of SEQ ID NO: 10. 5. An antibody or antigen-binding fragment according to any one of claims 1-4, which is multispecific. 6. An antibody or antigen binding fragment according to any one of claims 1 to 5, which includes one or more of the following properties: inhibits the growth of Plasmodium falciparum in human erythrocytes; inhibits the growth of Plasmodium falciparum strains D10, Dd2, 7G8, W2-mef, 3D7, HB3, FCR-1/FVO, Cam3.II or RF7 in human erythrocytes; binds to the PfRH5 polypeptide or an antigenic fragment thereof with a K _D of about 4.72 pM to about 1.67 nM at 25°C and/or from about 1.10 pM to about 1.10 nM at 37°C as measured by surface plasmon resonance; blocks the binding of the PfRH5 polypeptide to the basigin polypeptide; binds to PfRH5, which does not have amino terminal residues M1-Y139 and contains residues K140Q526, but does not contain K247-L295, and has mutations T216A and T299A; and/or causes a maximum inhibition of Plasmodium falciparum growth in heat-inactivated human or Aotus monkey serum that is about 1-10% greater than in non-heat-inactivated human or Aotus monkey serum, respectively; does not cause PfRH5 mutation in Plasmodium falciparum when exposed to the specified antigen-binding protein. 7. A complex containing an antibody or antigen-binding fragment according to any one of claims 1 to 6, associated with a Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) polypeptide. 8. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, including: (a) introducing one or more polynucleotides encoding an antibody or antigen binding fragment into a host cell; and (b) culturing the host cell under conditions favorable for expression of the one or more polynucleotides. 9. The method of claim 8, further comprising (c) isolating the antibody or antigen-binding fragment from the host cell and/or the medium in which the host cell is grown. 10. The method of claim 8 or 9, wherein the host cell is a Chinese hamster ovary cell. 11. Test strip for the presence of antigens for immunochromatographic analysis, containing an antibody or antigen-binding fragment according to any one of claims 1-6. 12. A polynucleotide encoding an antibody or antigen-binding fragment according to any one of claims 1 to 6. 13. A vector containing a polynucleotide according to claim 12. 14. A host cell containing the polynucleotide of claim 12 or the vector of claim 13. 15. A kit containing an antibody or antigen-binding fragment according to any one of claims 1 to 6. 16. The kit according to claim 15, further containing a therapeutic agent. 17. A pharmaceutical composition containing an antibody or antigen-binding fragment according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. 18. The pharmaceutical composition according to claim 17, further containing a therapeutic agent. 19. The composition or kit according to claim 16 or 18, wherein the additional therapeutic agent is an antiparasitic drug or vaccine. 20. The composition or kit according to any one of claims 15 to 19, characterized in that the additional therapeutic agent is a member selected from the group consisting of chloroquine, atovaquone, proguanil, artemether, lumefantrine, mefloquine, quinine, quinidine, doxycycline, clindamycin, vaccine and malaria vaccine. 21. A vessel or injection device containing an antibody or antigen-binding fragment according to any one of claims 1-6 or a composition according to any one of claims 17-20. 22. A method of treating or preventing Plasmodium falciparum infection in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen binding fragment according to any one of claims 1 to 6 or a pharmaceutical composition according to any one of claims 17 to 20. 23. The method of claim 22, wherein the subject has malaria or is at increased risk of contracting, or is predisposed to contracting malaria. 24. The method of claim 22 or 23, wherein the subject is diagnosed with Plasmodium falciparum infection before administering the antigen binding protein. 25. A method for diagnosing Plasmodium falciparum infection in a subject, comprising bringing into - 75 043756 contacting the antibody or antigen binding fragment according to any one of claims 1 to 6 with a sample from the specified subject and, if the sample shows a complex between the antibody or antigen binding fragment and the PfRH5 polypeptide, determining that the subject is infected with Plasmodium falciparum. 26. The method according to claim 25, where the specified complex is formed on a test strip for immunochromatographic analysis containing an antigen binding protein and a PfRH5 polypeptide. 27. A method of introducing an antibody or antigen-binding fragment according to any one of claims 1-6 into the body of a subject, comprising injecting the antibody or antigen-binding fragment into the body of the subject. 28. The method of claim 27, wherein the antibody or antigen binding fragment is administered subcutaneously, intravenously or intramuscularly to the subject.