Anti-CSP antibody variants

Abstract

The present disclosure provides anti-circumsporozoite (CSP) antibodies, compositions containing such antibodies, methods for producing the disclosed antibodies, and methods for using them to treat or prevent malaria, as well as methods for selecting antibodies as anti-malaria therapeutic antibodies.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 483,052, filed February 3, 2023, and U.S. Provisional Application No. 63 / 542,164, filed October 3, 2023, the entire contents of which are incorporated herein by reference for all purposes.

[0002] The present disclosure relates to compositions for treating or preventing malaria and to antibodies that confer protection against infection by insect-vector-transmitted malaria parasites, such as Plasmodium falciparum. The present disclosure also relates to methods for treating, preventing, or diagnosing Plasmodium infection in mammals. [Background technology]

[0003] Malaria causes a significant burden of morbidity and mortality, especially in developing countries. The causative agent of malaria is a protozoan parasite transmitted by mosquitoes. Several infectious Plasmodium species cause malaria, the most deadly of which is Plasmodium falciparum. Others include P. vivax, P. ovale, and P. malariae. More than 80% of malaria-related deaths occur in children under the age of five. RTS,S / AS01 (Mosquirix™), the most advanced vaccine and the only one recommended for use by the WHO, targets the circumsporozoite protein (CSP) of Plasmodium falciparum (Pf), the malaria species responsible for the leading cause of mortality in Africa. Three immunizations with RTS,S / AS01 induce anti-PfCSP antibodies, which act by binding to sporozoites, the infectious form of the malaria parasite introduced by mosquito bites, and inhibit their initial infection of liver cells. However, the immune response induced by Mosquirix™ in children is limited to 45% vaccine efficacy against clinical malaria after the first dose, decreasing to 36% over 4 years of follow-up. Therefore, other immunization approaches will be needed to achieve the WHO goal of reducing malaria case incidence and mortality by 90% by 2030.

[0004] Recent reports have shown that mAb treatment can completely prevent malaria after controlled infection and provide 88% efficacy (prevention of infection) for 6 months in endemic areas. Therefore, mAbs with durability lasting 4–6 months could provide an intervention with greater protective efficacy than that seen with RTS,S, greatly aiding efforts to prevent seasonal transmission. The mAbs tested in clinical trials, L911 and CIS438,9, isolated from B cells of vaccinees immunized with whole sporozoites, can prevent malaria infection by targeting specific epitopes on CSP. Given the published support for mAb prophylaxis as a strategy against malaria and the advances in the production of mAbs as therapeutic and prophylactic agents for common infectious diseases, there is a need to generate additional protective anti-CSP antibodies with improved therapeutic profiles. Summary of the Invention

[0005] The present disclosure provides anti-circumsporozoite (CSP) antibodies, compositions containing such antibodies, methods for producing the disclosed antibodies, and methods for using them to treat or prevent malaria, as well as methods for selecting antibodies as anti-malaria therapeutic antibodies.

[0006] In certain non-limiting embodiments, the present disclosure provides a recombinant anti-circumsporozoite (CSP) antibody that binds to a first epitope present in the central repeat region of CSP and to a second epitope of CSP. In certain embodiments, the first epitope comprises the amino acid sequence NPNA. In certain embodiments, the first epitope consists of an amino acid sequence selected from SEQ ID NOs: 923-974.

[0007] In certain embodiments, the second epitope is heterologous to an epitope present in the RTS,S vaccine. In certain embodiments, the second epitope comprises a CSP minor repeat region and / or a CSP junction region. In certain embodiments, the second epitope comprises a DPNA / NPNV-containing minor repeat amino acid sequence and / or a DPNA / NPNV-containing junction amino acid sequence. In certain embodiments, the second epitope consists of an amino acid sequence selected from SEQ ID NOs: 975-1195.

[0008] In certain embodiments, the antibody binds to at least one additional epitope of CSP. In certain embodiments, the at least one additional epitope comprises a DPNA / NPNV-containing minor repeat amino acid sequence and / or a DPNA / NPNV-containing junction amino acid sequence. In certain embodiments, the at least one additional epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975-1195.

[0009] In certain embodiments, the recombinant antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 80% identical to an amino acid sequence selected from SEQ ID NOs: 1 to 461. In certain embodiments, the recombinant antibody comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 461.

[0010] In certain embodiments, the recombinant antibody comprises a light chain variable region (VL) comprising an amino acid sequence at least about 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 462 to 922. In certain embodiments, the recombinant antibody comprises a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 462 to 922.

[0011] In certain embodiments, the recombinant antibody comprises a VH comprising an amino acid sequence at least about 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 461, and a VL comprising an amino acid sequence at least about 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 462 to 922. In certain embodiments, the recombinant antibody comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 461, and a VL comprising an amino acid sequence selected from SEQ ID NOs: 462 to 922.

[0012] In certain embodiments, the recombinant antibody comprises a heavy chain variable region (VH) and a light chain variable region (VH), wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the heavy chain variable sequence set forth in SEQ ID NOs: 1 to 461, and the light chain variable region comprises CDR1, CDR2, and CDR3 of the light chain variable sequence set forth in SEQ ID NOs: 462 to 922. In certain embodiments, the recombinant antibody comprises a heavy chain variable region (VH) and a light chain variable region (VH) set forth in Table 3.

[0013] In certain embodiments, the antibody exhibits at least a 20% reduction in parasite liver burden compared to the reference antibody. In certain embodiments, the antibody exhibits at least a 20% increase in survival rate compared to the reference antibody. In certain embodiments, the antibody exhibits increased conformational stability compared to the reference antibody. In certain embodiments, the antibody exhibits increased colloidal stability compared to the reference antibody. In certain embodiments, the reference antibody is AB-000317, AB-000224, or AB-007088.

[0014] In certain non-limiting embodiments, the present disclosure relates to polynucleotides encoding the antibodies of the present disclosure. In certain non-limiting embodiments, the present disclosure relates to host cells comprising the expression vectors and / or polynucleotides disclosed herein.

[0015] In certain non-limiting embodiments, the present disclosure also relates to compositions comprising the antibodies disclosed herein. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0016] In certain non-limiting embodiments, the present disclosure relates to methods of preventing or treating malaria in a subject in need thereof, comprising administering an effective amount of an antibody or composition disclosed herein, hi certain embodiments, the patient is a pediatric patient.

[0017] In certain non-limiting embodiments, the present disclosure relates to a method for selecting an antibody as an anti-malarial therapeutic antibody. In certain embodiments, the method comprises: a) analyzing the antibody for binding to a first epitope in the central repeat region of CSP; and b) analyzing the antibody for binding to a second epitope of CSP that is heterologous to the epitope present in the RTS,S vaccine, wherein the antibody is selected if it binds to both the first epitope and the second epitope. In certain embodiments, the method further comprises c) analyzing the antibody for binding to at least one additional epitope of CSP that is heterologous to the epitope present in the RTS,S vaccine, wherein the antibody is selected if it binds to the first epitope, the second epitope, and at least one additional epitope.

[0018] In certain non-limiting embodiments, the present disclosure relates to a method for selecting an antibody as an anti-malarial therapeutic antibody. In certain embodiments, the method comprises selecting an antibody if i) the antibody binds to a first epitope in the central repeat region of a CSP, and ii) the antibody binds to a second epitope heterologous to the epitope present in the RTS,S vaccine. In certain embodiments, the method comprises selecting an antibody if i) the antibody binds to a first epitope in the central repeat region of a CSP, ii) the antibody binds to a second epitope heterologous to the epitope present in the RTS,S vaccine, and iii) the antibody binds to at least one additional epitope heterologous to the epitope present in the RTS,S vaccine.

[0019] In certain embodiments, the first epitope comprises the amino acid sequence NPNA. In certain embodiments, the first epitope consists of an amino acid sequence selected from SEQ ID NOs: 923-974. In certain embodiments, the second epitope is heterologous to an epitope present in the RTS,S vaccine. In certain embodiments, the second epitope comprises a CSP minor repeat region and / or a CSP junction region. In certain embodiments, the second epitope comprises a DPNA / NPNV-containing minor repeat amino acid sequence and / or a DPNA / NPNV-containing junction amino acid sequence. In certain embodiments, the second epitope consists of an amino acid sequence selected from SEQ ID NOs: 975-1195. In certain embodiments, the first epitope consists of an amino acid sequence selected from SEQ ID NOs: 923-974 and the second epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975-1195.

[0020] In certain embodiments, the antibody has a binding affinity to the first epitope of about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 A binding affinity (K D In certain embodiments, the antibody binds to the second epitope at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D In certain embodiments, the antibody binds to the first epitope at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D and binds to a second epitope at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D Combine with.

[0021] In certain embodiments, a) the first epitope consists of an amino acid sequence selected from SEQ ID NOs: 923-974, b) the second epitope consists of an amino acid sequence selected from SEQ ID NOs: 975-1195, and c) at least one additional epitope consists of an amino acid sequence selected from SEQ ID NOs: 975-1195.

[0022] In certain embodiments, the antibody has about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is less than M D In certain embodiments, the antibody binds to a first epitope at a binding affinity of about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D and b) binds to a second epitope at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D and c) binds to at least one additional epitope at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D Combine with. [Brief explanation of the drawings]

[0023] [Figure 1A] Functional antibodies bind to CSP-derived peptides not present in the RTS,S vaccine. Percent inhibition and number of germline-derived nucleotide mutations in a sporozoite liver-challenged mouse model are shown for mAbs reactive to the NANP6 repeat region peptide (circles, n = 67) or the C-terminal region peptide (squares, n = 10), shown as protected (green) or unprotected (blue), and from vaccinees receiving standard (012M, closed symbols) or split (Fx017M, open symbols) doses. For correlation of untransformed data, P < 0.03 for all comparisons (Spearman test) and P < 0.04 for all comparisons (Pearson test) except for NVDP3NANP2, where P > 0.5. [Figure 1B]Functional antibodies bind to CSP-derived peptides not present in the RTS,S vaccine. SPR-determined binding capacities (KDs) of antibodies (n=141) selected from a panel of 35 of the most effective strains tested against CSP and CSP-derived peptides that are homologous (NANP6, NPNA3) or heterologous (NVDP3NANP2, NPDPNANPNVDPNANP, conjugates) to RTS,S are shown. Examples are shown of an antibody with a broad, promiscuous binding profile (green, AB-007163), an antibody with a profile relatively biased toward homologous peptides (purple, AB-007143), and an antibody with a profile between these extremes (blue, AB-007175). For correlations of untransformed data, P<0.03 (Spearman test) for all comparisons and P<0.04 (Pearson test) for all comparisons except for NVDP3NANP2 (P>0.5). [Figure 1C] Functional antibodies bind to CSP-derived peptides not present in the RTS,S vaccine. Linear regression comparing the number of germline-derived nucleotide mutations (SHM) per heavy chain versus the log-transformed SPR binding off-rate (koff) for peptide is shown, with short major repeats (NPNA3, n=140). For correlation of untransformed data, P<0.03 for all comparisons (Spearman test) and P<0.04 for all comparisons except for NVDP3NANP2 (P>0.5) (Pearson test). [Figure 1D] Functional antibodies bind to CSP-derived peptides not present in the RTS,S vaccine. Linear regression comparing the number of germline-derived nucleotide mutations (SHM) per heavy chain versus the log-transformed SPR binding off-rate (koff) for peptides is shown, with conjugation (KQPADGNPDPNANPN, n=68). For correlation of untransformed data, P<0.03 (Spearman test) for all comparisons and P<0.04 (Pearson test) for all comparisons except for NVDP3NANP2, P>0.5. [Figure 1E]Functional antibodies bind to CSP-derived peptides not present in the RTS,S vaccine. Linear regression comparing the number of germline-derived nucleotide mutations (SHM) per heavy chain versus the log-transformed SPR binding off-rate (koff) for peptides is shown, with short minor repeats (NPDPNANPNVDPNANP, n=109). For correlation of untransformed data, P<0.03 (Spearman test) for all comparisons and P<0.04 (Pearson test) for all comparisons except for NVDP3NANP2, where P>0.5. [Figure 1F] Functional antibodies bind to CSP-derived peptides not present in the RTS,S vaccine. Linear regression comparing the number of germline-derived nucleotide mutations (SHM) per heavy chain versus the log-transformed SPR binding off-rate (koff) for peptides is shown, with the long major repeat (NANP6, n=141). For correlation of untransformed data, P<0.03 (Spearman test) for all comparisons and P<0.04 (Pearson test) for all comparisons except for NVDP3NANP2, P>0.5. [Figure 1G] Functional antibodies bind to CSP-derived peptides not present in the RTS,S vaccine. Linear regression comparing the number of germline-derived nucleotide variations (SHM) per heavy chain versus the log-transformed SPR binding off-rate (koff) for the peptide, long minor repeat (NVDP3NANP2, n=129), is shown. For correlation of untransformed data, P<0.03 (Spearman test) for all comparisons and P<0.04 (Pearson test) for all comparisons except for NVDP3NANP2, P>0.5. [Figure 2A] Figure 1 shows in vivo pharmacology, SHM, and binding of anti-CSP antibodies. Data from a liver burden model are shown, showing percent inhibition of n=69 antibodies (32 lines) compared to AB-000317; non-gray colors indicate the six lines containing the most effective antibodies (*P>0.05, †P<0.05, two-tailed, non-parametric log-rank). [Figure 2B]In vivo pharmacology, SHM, and anti-CSP antibody binding are shown. Data from the liver burden model are shown. Parasite bioluminescence in the liver (total flux, photons / second). *P<0.035 for AB-000224 and AB-000317. Lines and bars indicate the geometric mean and geometric standard deviation. P>0.2 (ns), two-tailed Mann-Whitney test. [Figure 2C] In vivo pharmacology, SHM, and binding of anti-CSP antibodies. Data from the liver challenge model are shown; *P<0.035 and serum concentrations of antibodies (serum [Ab], μg / ml) at the time of sporozoite challenge (AB-000224 and AB-000317). Lines and bars indicate geometric means and geometric standard deviations; P>0.2 (ns), two-tailed Mann-Whitney test. [Figure 2D] In vivo pharmacology, SHM, and binding of anti-CSP antibodies are shown. Percent liver burden inhibitory activity normalized to AB-000317 is shown, with each antibody indicated as having significantly greater (dark blue triangles), no difference (green circles), or weaker (light blue triangles) activity than AB-000317 (two-sided, non-parametric log-rank) binding off-rates (SPR, koff) against the CSP (n=70) peptide. [Figure 2E] In vivo pharmacology, SHM, and binding of anti-CSP antibodies are shown. Percent liver burden inhibitory activity normalized to AB-000317 is shown, with each antibody indicated as having significantly greater activity than AB-000317 (dark blue triangles), no difference (green circles), or weaker activity (light blue triangles) (two-sided, non-parametric log-rank) versus binding off-rates (SPR, koff) for the major replicate (NPNA3, n=70) peptide. [Figure 2F]In vivo pharmacology, SHM, and binding of anti-CSP antibodies are shown. Percent liver load inhibitory activity normalized to AB-000317 is shown, with each antibody indicated as having significantly greater (dark blue triangles), no difference (green circles), or weaker (light blue triangles) activity than AB-000317 (two-sided, non-parametric log-rank) and binding off-rates (SPR, koff) for the conjugated (KQPADGNPDPNANPN, n=42) peptide. [Figure 2G] In vivo pharmacology, SHM, and binding of anti-CSP antibodies are shown. Percent liver burden inhibitory activity normalized to AB-000317 is shown, with each antibody indicated as having significantly greater (dark blue triangles), no difference (green circles), or weaker (light blue triangles) activity than AB-000317 (two-sided, non-parametric log-rank) binding off-rates (SPR, koff) for the minor repeat (NPDPNANPNVDPNANP, n=60) peptide. [Figure 2H] Figure 1 shows the in vivo pharmacology, SHM, and binding of anti-CSP antibodies. Percent liver load inhibitory activity normalized to AB-000317 is shown, with each antibody indicated as having significantly greater activity than AB-000317 (dark blue triangles), no difference (green circles), or weaker activity (light blue triangles) (two-sided, nonparametric log-rank analysis). For each antibody (n=70), the number of amino acid residue changes from SHM in the heavy chain is shown. Linear regression of log-transformed data is shown. For correlation of untransformed data, P<0.005 for all comparisons (Pearson test) and P<0.05 for all comparisons except for P>0.06 for liver load inhibition vs. koff[NPDPNANPNVDPNANP] (Spearman test). [Figure 2I]Figure 1 shows the in vivo pharmacology, SHM, and binding of anti-CSP antibodies. Percent liver load inhibitory activity normalized to AB-000317 is shown, with each antibody indicated as having significantly greater activity than AB-000317 (dark blue triangles), no difference (green circles), or weaker activity (light blue triangles) (two-sided, nonparametric log-rank analysis). For each antibody (n=70), the number of amino acid residue changes from SHM in the light chain is shown. Linear regression of log-transformed data is shown. For correlation of untransformed data, P<0.005 for all comparisons (Pearson test) and P<0.05 for all comparisons except for P>0.06 for liver load inhibition vs. koff[NPDPNANPNVDPNANP] (Spearman test). [Figure 2J] In vivo pharmacology, SHM, and binding of anti-CSP antibodies are shown. SPR binding off-rates (k) versus on-rates (k) for the NPNA3 peptide are shown for antibodies with high SHM (green, ≥20 mutations per clone, n = 56) or low SHM (blue, <20 mutations per clone, n = 14), and weaker (downward triangles), no difference (circles), or better (upward triangles) activity than AB-000317 (two-sided, nonparametric log-rank). Monoclonal antibodies from lines reported to bind CSP with Fab-Fab homotypic interactions are shown (red circles, AB-00039942, AB-007159, AB-007160, AB-007161). [Figure 2K] In vivo pharmacology, SHM, and binding of anti-CSP antibodies. Hazard ratios for n=25 antibodies (14 strains) compared to AB-000317 in a mosquito bite parasitemia model are shown. Non-gray colors indicate the five strains containing the most effective antibodies. [Figure 2L] In vivo pharmacology, SHM, and binding of anti-CSP antibodies are shown. Survival curves from replicate experiments comparing AB-000224 [0.74 (0.15, 3.8)] to AB-000317, two-sided, nonparametric log-rank [Mantel-Haenszel hazard ratio (95% confidence interval)] are shown. [Figure 2M] In vivo pharmacology, SHM, and binding of anti-CSP antibodies are shown. Survival curves from replicate experiments comparing AB-007088 [0.61 (0.097, 3.8)] to AB-000317, two-sided, nonparametric log-rank [Mantel-Haenszel hazard ratio (95% confidence interval)] are shown. [Figure 3A] CSP-reactive lineages from blood PBs after the third dose of RTS,S are shown. IgG lineages from each vaccinee (bars, n = 45) are indicated by the number of lineages: clonally expanded (i.e., with two or more distinct nucleotide clones, green); cellularly expanded but with only one IgG clone observed (i.e., with two or more identical nucleotide clones, gray); or no evidence of recent expansion and containing only one observed PB (blue). Lineages from which clonality was selected for testing (n = 369) are indicated by CSP reactivity: CSP-reactive (green dots, n = 139), indeterminate (orange dots, n = 29), or non-reactive (blue triangles, n = 201). All lineages not tested are shown (gray circles, n = 13,134, with 2,313 and 10,821 single PB lineages expanded). Protected vaccinees had a lower ratio of CSP-reactive to non-reactive lineages than unprotected vaccinees (bootstrap analysis, P = 0.0011). The red circles indicate the two lines containing the amino acid sequence of AB-000317. [Figure 3B]CSP-reactive lineages from blood PBs after the third dose of RTS,S are shown. IgG lineages from each vaccinee (bars, n = 45) are shown by the number of PBs per vaccinee. They were clonally expanded (i.e., had two or more distinct nucleotide clones, green), cellularly expanded but only one IgG clone was observed (i.e., had two or more identical nucleotide clones, gray), or contained only one observed PB (blue) without evidence of recent expansion. Lineages from which clonality was selected for testing (n = 369) are indicated by CSP reactivity: CSP-reactive (green dots, n = 139), indeterminate (orange dots, n = 29), or not reactive (blue triangles, n = 201). All lineages not tested are shown (gray circles, n = 13,134, with 2,313 expanded and 10,821 single PB lineages). Protected vaccinees had a lower ratio of CSP-reactive to non-reactive strains than unprotected vaccinees (bootstrap analysis, P=0.0011). Red circles indicate two strains containing the amino acid sequence of AB-000317. [Figure 3C]CSP-reactive lineages derived from blood PBs after the third dose of RTS,S are shown. For each vaccinee, the size of each expanded lineage was calculated by dividing the number of PBs in that lineage by the number of PBs in all expanded lineages within each repertoire and then assigning a rank size. Boxes indicate the interquartile range, the line within the box is the median, and the whiskers represent the minimum and maximum values ​​across vaccinees for each rank size. The lineages with the top four rank sizes contain 33% of the PBs in all expanded lineages (dotted lines). Lineages from which clones were selected for testing (n = 369) are indicated by CSP reactivity: CSP-reactive (green dots, n = 139), indeterminate (orange dots, n = 29), or not reactive (blue triangles, n = 201). All lineages not tested are shown (gray circles, n = 13,134, 2,313 expanded and 10,821 single-PB lineages). Protected vaccinees had a lower ratio of CSP-reactive to non-reactive strains than unprotected vaccinees (bootstrap analysis, P=0.0011). Red circles indicate two strains containing the amino acid sequence of AB-000317. [Figure 3D]CSP-reactive lineages derived from blood PB after the third dose of RTS,S are shown. ELISA reactivity, SHM levels, and vaccinee protection status of mAbs derived from the expanded lineages (n=349) are shown. The number of germline-derived nucleotide mutations (SHM) is shown for mAbs that were not reactive (dark blue, n=185), showed an indeterminate or weak signal (orange, n=29), or were reactive (light green, n=135) in the CSP ELISA. Domain specificity for CSP-reactive mAbs is indicated in light green boxes. Monoclonal antibodies reactive by ELISA to the NANP6 repeat region peptide (light green, n=98), the C-terminal region peptide (Pfs16, light blue, n=20), or not reactive to either peptide ELISA (light green, n=9). Lines represent medians; ***P<0.0001, **P<0.001, unpaired two-tailed Mann-Whitney test. CSP-reactive mAbs (n=8) not tested by peptide ELISA are not shown. Strains (n=369) from which clones were selected for testing are indicated by CSP reactivity: CSP-reactive (green dots, n=139), indeterminate (orange dots, n=29), or non-reactive (blue triangles, n=201). All strains not tested are shown (gray circles, n=13,134, 2,313 and 10,821 expanded single PB strains). Protected vaccinees had a lower ratio of CSP-reactive to non-reactive strains than unprotected vaccinees (bootstrap analysis, P=0.0011). Red circles indicate two strains containing the amino acid sequence of AB-000317. [Figure 3E]CSP-reactive lines from blood PB after the third dose of RTS,S are shown. ELISA reactivity, SHM levels, and vaccinee protection status (n=349) of mAbs from expanded lines are shown. Percentages of tested antibodies from expanded lines from protected (green, n=36) and unprotected (blue, n=9) vaccinees are shown, including CSP reactivity (82 / 249 and 53 / 100 mAbs, respectively) and repeat regions. **P<0.001, *P<0.01, Fisher's exact test. Lines from which clones were selected for testing (n=369) are indicated by CSP reactivity: CSP-reactive (green dots, n=139), indeterminate (orange dots, n=29), or non-reactive (blue triangles, n=201). All lines not tested are shown (gray circles, n=13,134, 2,313 expanded, and 10,821 single PB lines). Protected vaccinees had a lower ratio of CSP-reactive to non-reactive strains than unprotected vaccinees (bootstrap analysis, P=0.0011). Red circles indicate two strains containing the amino acid sequence of AB-000317. [Figure 3F]CSP-reactive lines from blood PB after the third dose of RTS,S are shown. ELISA reactivity, SHM levels, and vaccinee protection status of mAbs from expanded lines (n=349). The percent of tested antibodies from expanded lines from protected (green, n=36) and unprotected (blue, n=9) vaccinees that were reactive to the repeat region and NANP6 peptide (59 / 244 and 39 / 97 mAbs, respectively) are shown. **P<0.001, *P<0.01, Fisher's exact test. Lines from which clones were selected for testing (n=369) are indicated by CSP reactivity: CSP-reactive (green dots, n=139), indeterminate (orange dots, n=29), or non-reactive (blue triangles, n=201). All lines not tested are shown (gray circles, n=13,134, 2,313 expanded, and 10,821 single PB lines). Protected vaccinees had a lower ratio of CSP-reactive to non-reactive strains than unprotected vaccinees (bootstrap analysis, P=0.0011). Red circles indicate two strains containing the amino acid sequence of AB-000317. [Figure 3G]CSP-reactive lineages from blood PB after the third dose of RTS,S are shown. ELISA reactivity, SHM levels, and vaccinee protection status of mAbs from expanded lineages (n=349). The percentage of tested antibodies from expanded lineages derived from protected (green, n=36) and unprotected (blue, n=9) vaccinees is shown, which is a subset of only the dominant rank size 1-4 lineages that are repeat and CSP-reactive (52 / 142 and 31 / 46 mAbs, respectively). **P<0.001, *P<0.01, Fisher's exact test. Lineages (n=369) from which clones were selected for testing are indicated by CSP reactivity: CSP-reactive (green dots, n=139), indeterminate (orange dots, n=29), or non-reactive (blue triangles, n=201). All strains not tested are shown (gray circles, n=13,134, 2,313 propagated and 10,821 single PB strains). Protected vaccinees had a lower ratio of CSP-reactive to non-reactive strains than unprotected vaccinees (bootstrap analysis, P=0.0011). Red circles indicate two strains containing the amino acid sequence of AB-000317. [Figure 3H] CSP-reactive lineages from blood PB after the third dose of RTS,S are shown. For vaccinees indicated on the x-axis, each symbol represents a single lineage. Lineages (n=369) from which clones were selected for testing are indicated by CSP reactivity: CSP-reactive (green dots, n=139), indeterminate (orange dots, n=29), or non-reactive (blue triangles, n=201). All lineages not tested are shown (gray circles, n=13,134, 2,313 and 10,821 single PB lineages expanded). Protected vaccinees had a lower ratio of CSP-reactive to non-reactive lineages than unprotected vaccinees (bootstrap analysis, P=0.0011). Red circles represent two lineages containing the amino acid sequence of AB-000317. [Figure 4]Shown is a histogram of the number of germline-derived nucleotide mutations (SHM) in combined IgG heavy and light chains from blood PB collected 7 days after administration of the third dose (blue, n = 22,319) or fourth dose (gray, n = 10,429) of RTS,S. [Figure 5A] IgG sequence and repertoire characteristics of PB responses after the third dose of RTS,S are shown. For heavy chains, germline V gene usage is compared between protected (green, n=36) and unprotected (blue, n=9) subjects and between dose groups (standard dose group, "012M," n=15; split dose group, "Fx017M," n=30). IGHV3-30, IGHV3-33, KV1-5, KV3-20, and LV1-40 show high prevalence. Boxes indicate interquartile ranges; the line within the box is the median; whiskers represent the furthest data point within 1.5 times the interquartile range; points outside the whiskers are plotted individually as outliers. [Figure 5B] IgG sequence and repertoire characteristics of PB responses after the third dose of RTS,S are shown. For light chains, germline V gene usage is compared between protected (green, n=36) and unprotected (blue, n=9) subjects and between dose groups (standard dose group, "012M," n=15; split dose group, "Fx017M," n=30). IGHV3-30, IGHV3-33, KV1-5, KV3-20, and LV1-40 show high prevalence. Boxes indicate interquartile ranges; the line within the box is the median; whiskers represent the furthest data point within 1.5 times the interquartile range; points outside the whiskers are plotted individually as outliers. [Figure 5C]IgG sequence and repertoire characteristics of PB responses after the third dose of RTS,S are shown. Germline V gene usage for heavy chain (A) and light chain (B) is compared between protected (green, n=36) and unprotected (blue, n=9) subjects and between dose groups (standard dose group, "012M," n=15; split dose group, "Fx017M," n=30). IGHV3-30, IGHV3-33, KV1-5, KV3-20, and LV1-40 show high prevalence. Specific pairings of heavy and light chain genes are shown. Boxes indicate the interquartile range; the line within the box is the median; whiskers represent the furthest data point within 1.5 times the interquartile range; points outside the whiskers are plotted individually as outliers. [Figure 5D] IgG sequence and repertoire characteristics of PB responses after the third dose of RTS,S are shown. Three heavy chain germline genes, IGHV3-73, IGHV4-61, and IGHV5-51, were initially associated with vaccinee protection status (P<0.05, Wilcoxon rank-sum test) but not after correcting for multiple hypothesis testing. All P>0.05, Benjamini-Hochberg or Bonferroni tests. Boxes indicate interquartile ranges; the line within the box is the median; whiskers represent the furthest data point within 1.5 times the interquartile range; points outside the whiskers are plotted individually as outliers. [Figure 5E] IgG sequence and repertoire characteristics of PB responses after the third dose of RTS,S are shown. No significant associations were detected between vaccinee protection status and dose group for IgG heavy and light chain constant region subclasses (P>0.05 for all analyses, Wilcoxon rank sum test). Boxes indicate interquartile ranges; the line within the box is the median; whiskers represent the furthest data point within 1.5 times the interquartile range; points outside the whiskers are plotted individually as outliers. [Figure 5F]IgG sequence and repertoire characteristics of PB responses after the third dose of RTS,S are shown. No significant associations were detected between vaccinee protection status and dose group for IgG heavy and light chain constant region subclasses (P>0.05 for all analyses, Wilcoxon rank sum test). Boxes indicate interquartile ranges; the line within the box is the median; whiskers represent the furthest data point within 1.5 times the interquartile range; points outside the whiskers are plotted individually as outliers. [Figure 5G] IgG sequence and repertoire characteristics of the PB response after the third dose of RTS,S are shown. For analyses including lineages with only one PB, no significant association was detected between vaccinee protection status and dose group for repertoire clonality (normalized Shannon entropy, P > 0.05 for all analyses, Wilcoxon rank sum test or Kolmogorov-Smirnov test). Boxes indicate the interquartile range; the line within the box is the median; whiskers represent the furthest data point within 1.5 times the interquartile range; points outside the whiskers are plotted individually as outliers. [Figure 5H] IgG sequence and repertoire characteristics of PB responses after the third dose of RTS,S are shown. No significant association was detected between vaccinee protection status and dose group for heavy chain complementarity-determining region 3 length (CDR3; P > 0.05 for both, Wilcoxon rank-sum test). Boxes indicate interquartile ranges; the line within the box is the median; whiskers represent the furthest data point within 1.5 times the interquartile range; points outside the whiskers are plotted individually as outliers. [Figure 5I] IgG sequence and repertoire characteristics of PB responses after the third dose of RTS,S are shown. No significant association was detected between vaccinee protection status and dose group for light chain complementarity-determining region 3 length (CDR3; P > 0.05 for both, Wilcoxon rank-sum test). Boxes indicate interquartile ranges; the line within the box is the median; whiskers represent the furthest data point within 1.5 times the interquartile range; points outside the whiskers are plotted individually as outliers. [Figure 6A]Antibody lineages tested in binding assays and reactivity to CSP or HBsAg are shown. Rank sizes of expanded lineages in the repertoires of each protected and unprotected vaccinee from PB collected 7 days after the third dose of RTS,S are shown. For each rank size and vaccinee, expanded PB antibody lineages (circles representing one or more lineages) are shown from which mAbs were selected, recombinantly expressed, and screened in the CSP ELISA (n = 349 mAbs, 282 circles). Circle size is proportional to the proportion of lineages tested among all lineages observed for each rank size and vaccinee. Lineages from vaccinees with the same number of PBs have the same rank size. The largest circle indicates that all lineages from vaccinees of that rank size were tested. The smallest circle indicates that only 1 of the 58 lineages observed from vaccinees at that rank size were tested. In some cases, none of the lineages from vaccinees of the indicated rank size were tested (gray bars). [Figure 6B] Antibody lineages tested in binding assays and reactivity to CSP or HBsAg are shown. Rank size of expanded lineages in the repertoires of each protected and unprotected vaccinee from PB collected 7 days after the third dose of RTS,S. For each rank size and vaccinee, expanded PB antibody lineages (circles representing lineages 1–5) are shown, from which mAbs were tested in the CSP ELISA: reactive (green, n=135 mAb, 94 circles), equivocal (gray, n=29 mAb, 14 circles), non-reactive (blue, n=185 mAb, 144 circles), or a combination of these results for different mAbs from the same rank size and vaccinee lineage (mixed-color pie chart, 30 circles). [Figure 6C]Antibody lineages tested in binding assays and reactivity to CSP or HBsAg are shown. Rank size of expanded lineages in the repertoires of each protected and unprotected vaccinee from PB collected 7 days after the third dose of RTS,S. For each rank size and vaccinee, expanded PB antibody lineages (circles representing lineages 1–5) are shown, from which mAbs were tested in HBsAg ELISA: reactive (green, n=38 mAbs, 36 circles), equivocal (gray, n=3 mAbs, 3 circles), non-reactive (blue, n=77 mAbs, 72 circles), or a combination of these results for different mAbs from the same rank size and vaccinee lineage (mixed-color pie chart, 2 circles). [Figure 7A] SHM and CSP peptide binding of mAbs versus RTS,S dose group and vaccinee protection status are shown. Distribution of SHM levels of mAbs from expanded lines of vaccinees receiving either the third standard dose (012M, dark blue, n=15) or the delayed split dose (Fx017M, orange, n=30) of RTS,S. mAbs reactive to the CSP repeat region (NANP6, n=45 and n=53, respectively), the C-terminal region of CSP (C-terminus, n=4 and n=16, respectively), or not reactive to either peptide (negative, n=5 and n=4, respectively) compared to mAbs not reactive in the CSP ELISA (n=72 and n=113, respectively). [Figure 7B]SHM and CSP peptide binding of mAbs versus RTS,S dose group and vaccinee protection status are shown. Distribution of SHM levels for mAbs derived from the expanded lineages of vaccinees from protected (green, n = 36) or unprotected (light blue, n = 9) vaccinees. mAbs reactive to the CSP repeat region (n = 59 and n = 39, respectively), the C-terminal region of CSP (n = 12 and n = 8, respectively), or not reactive to either peptide (negative, n = 6 and n = 3, respectively) are shown compared to mAbs not reactive in the CSP ELISA (n = 147 and n = 38, respectively). Lines indicate medians; ***P < 0.0001, **P < 0.001, *P < 0.02, or P > 0.1 ("ns"), unpaired, two-tailed Mann-Whitney test. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide those skilled in the art with a general guide to many of the terms used in this application.

[0026] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Thus, for example, reference to a "protein" or an "antibody" includes a plurality of proteins or antibodies, respectively, reference to a "cell" includes a mixture of cells, and the like.

[0027] As used herein, the term "about" or "approximately" refers to the normal error range for the respective value, readily known to one of ordinary skill in the art, e.g., ±20%, ±10%, or ±5% within the intended meaning of the recited value.

[0028] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that contains the necessary variable region sequence for specific binding to an antigen epitope. Thus, as used herein, "antibody" refers to any form of antibody or fragment thereof that exhibits the desired biological activity, such as binding to a specific target antigen. Thus, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including, but not limited to, scFv, Fab, etc., as long as they exhibit the desired biological activity.

[0029] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies (e.g., Zapata et al., Protein Eng. 8(10):1057-1062(1995)), single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, a name reflecting their ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0030] As used herein, the terms "anti-CSP antibody" and "CSP antibody" are used interchangeably and refer to an antibody that binds to the circumsporozoite (CSP) antigen of Plasmodium falciparum.

[0031] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay.

[0032] As used herein, "V region" refers to an antibody variable region domain comprising framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4 segments. The heavy chain V region, VH, is the result of rearrangement of the V gene (HV), D gene (HD), and J gene (HJ), known as V(D)J recombination, during B cell differentiation. The light chain V region, VL, is the result of rearrangement of the V gene (LV) and J gene.

[0033] As used herein, "complementarity-determining region (CDR)" refers to the three hypervariable regions (HVRs) in each chain that interrupt the four "framework" regions established by the light and heavy chain variable regions. CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of each chain are designated CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus and identified by the chain in which the particular CDR is located. Thus, a VH CDR3 (HCDR3) is located in the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR3 (LCDR3) is the CDR3 from the variable domain of the light chain of the antibody in which it is found. The term "CDR" is used interchangeably with "HVR" when referring to a CDR sequence.

[0034] The amino acid sequences of the CDRs and framework regions can be determined using various definitions in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT), and AbM (see, for example, Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, Structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol. 1997, 273(4)).The definition of antigen binding sites is also described in: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000), and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1; 29(1): 207-9 (2001); MacCallum et al., Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262(5), 732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al., Methods Enzymol., 203, 121-153 (1991); Pedersen et al. al, Immunomethods, 1,126, (1992), and Rees et al, In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996). References to CDRs determined by Kabat numbering are based on, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Chothia CDRs are determined as defined by Chothia (see, for example, Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).CDRs can also be determined using available in silico systems as described in Swindells et al., Journal of molecular biology 429.3(2017):356-364, the contents of which are incorporated by reference in their entirety.

[0035] "Fc region" refers to the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. In the case of IgA and IgM, Fc may include the J chain. In the case of IgG, Fc includes immunoglobulin domains Cy2 and Cy3 and the hinge between Cy1 and Cy2. Although the boundaries of the Fc region may vary in the art, the human IgG heavy chain Fc region is usually understood to be defined as including residues C226 or P230 at its carboxy terminus, using numbering according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The term "Fc region" can refer to this region in isolation or in the context of an antibody or antibody fragment. "Fc region" includes naturally occurring allelic variants of the Fc region and modifications that modulate effector function. The Fc region also includes variants that do not result in altered biological function. For example, one or more amino acids can be deleted from the N- or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art to have minimal effect on activity (see, e.g., Bowie, et al., Science 247:306-1310, 1990). For example, for an IgG4 antibody, a single amino acid substitution (S228P according to Kabat numbering, designated IgG4Pro) can be introduced to eliminate the heterogeneity observed in recombinant IgG4 antibodies (see, e.g., Angal, et al., Mol Immunol 30:105-108, 1993). In certain embodiments, the Fc region comprises a substitution that improves the pharmacokinetic properties of the antibody, e.g., increased serum half-life.Non-limiting examples of Fc region substitutions can be found in US Pat. No. 8,088,376, the contents of which are incorporated by reference in their entirety.

[0036] The term "equilibrium dissociation constant", abbreviated as (KD), is the dissociation rate constant (kd, time -1 ) is the association rate constant (ka, time -1 M -1 ) divided by 10. Any method can be used to measure the equilibrium dissociation constant. Thus, in certain embodiments, antibodies of the present disclosure have a KD of less than about 50 nM, typically less than about 25 nM or less than 10 nM, e.g., less than about 5 nM, or less than about 1 nM, and often less than about 10 nM, as determined by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37°C. In certain embodiments, antibodies of the present disclosure have a KD of less than 5 x 10 when measured as a bivalent antibody. -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10 -9 Less than M, 5 x 10 -10 Under M, 10 -10 Less than M, 5 x 10 -11M Less than 10 -11 Less than M, 5 x 10 -12 Under M, 10 -12 Less than M, 5 x 10 -13 Under M, 10 -13 Less than M, 5 x 10 -14 Under M, 10 -14 Less than M, 5 x 10 -15 Less than M or 10 -15 As used herein, an "improved" KD refers to a lower KD. In certain embodiments, the antibodies of the present disclosure have a KD of 5 x 10 when measured as a monovalent antibody, such as a monovalent Fab. -5 Under M, 10-5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10 -9 Less than M, 5 x 10 -10 Under M, 10 -10 Less than M, 5 x 10 -11 Under M, 10 -11 Less than M, 5 x 10 -12 Under M, 10 -12 Less than M, 5 x 10 -13 Under M, 10 -13 Less than M, 5 x 10 -14 Under M, 10 -14 Less than M, 5 x 10 -15 Less than M or 10 -15 In certain embodiments, an anti-CSP antibody of the present disclosure has a KD of less than 100 pM, e.g., less than 75 pM, e.g., in the range of 1 to 100 pM, when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37° C. In certain embodiments, an anti-CSP antibody of the present disclosure has a KD of more than 100 pM, e.g., in the range of 100 to 1000 pM or 200 to 1000 pM, when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37° C.

[0037] The term "monovalent molecule" as used herein refers to a molecule that has one antigen-binding site, e.g., a Fab or scFv.

[0038] As used herein, the term "bivalent molecule" refers to a molecule having two antigen-binding sites. In certain embodiments, a bivalent molecule of the present invention is a bivalent antibody or a bivalent fragment thereof. In certain embodiments, a bivalent molecule of the present invention is a bivalent antibody. In certain embodiments, a bivalent molecule of the present invention is an IgG. In certain embodiments, a monoclonal antibody has a bivalent basic structure. IgG and IgE have only one bivalent unit, while IgA and IgM consist of multiple bivalent units (two and five, respectively) and therefore have a higher valency. This bivalency increases the avidity of the antibody for the antigen.

[0039] As used herein, the terms "monovalent binding" or "monovalently bind" refer to binding of one antigen-binding site to the antigen.

[0040] As used herein, the terms "bivalent binding" or "bivalently bind" refer to both antigen-binding sites of a bivalent molecule binding to the antigen. In certain embodiments, both antigen-binding sites of a bivalent molecule share the same antigen specificity.

[0041] As used herein, the term "valency" refers to the number of different binding sites of an antibody for an antigen. A monovalent antibody contains one binding site for an antigen. A bivalent antibody (e.g., a bivalent IgG antibody) contains two binding sites for the same antigen.

[0042] The term "affinity," as used herein, refers to either the single or combined strength of one or both arms of an antibody (e.g., an IgG antibody) binding to one or more epitopes expressed on either a simple or complex antigen. As defined herein, the term "affinity" does not imply a particular valency between two binding partners.

[0043] The phrases "specifically (or selectively) bind" to an antigen or target, or "specifically (or selectively) immunoreactive" when referring to a protein or peptide, refer to a binding reaction whereby an antibody binds to the antigen or target of interest with an affinity that is distinguishable from non-specific interactions that occur between two proteins.

[0044] In the context of two or more polypeptide sequences, the term "identical" or percent "identity" refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence over a comparison window or designated region, are the same or have a specified percentage of identical amino acid residues (e.g., at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) over a particular region, e.g., the length of the two sequences. Alignment for purposes of determining percent amino acid sequence identity can be performed in a variety of ways, including, but not limited to, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which is described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). In certain embodiments, percent sequence identity can be determined using BLAST 2.0 with default parameters.

[0045] As used herein, a "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

[0046] As used herein, "conservative" substitution refers to the substitution of amino acids such that the charge, polarity, hydropathy (hydrophobic, neutral, or hydrophilic), and / or size of the side group chain are maintained. Exemplary sets of amino acids that can be substituted for each other include: (i) the positively charged amino acids Lys and Arg, and His at a pH of about 6; (ii) the negatively charged amino acids Glu and Asp; (iii) the aromatic amino acids Phe, Tyr, and Trp; (iv) the nitrogen ring amino acids His and Trp; (v) the aliphatic hydrophobic amino acids Ala, Val, Leu, and Ile; (vi) the hydrophobic sulfur-containing amino acids Met and Cys, which are less hydrophobic than Val, Leu, and Ile; (vii) the small polar uncharged amino acids Ser, Thr, Asp, and Asn; (viii) the small hydrophobic or neutral amino acids Gly, Ala, and Pro; (ix) the amide-containing amino acids Asn and Gln; and (xi) the beta-branched amino acids Thr, Val, and Ile. References to the charge of an amino acid refer to the charge at pH 6-7.

[0047] As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to both the sense and antisense strands of RNA, cDNA, genomic DNA, and the above synthetic forms and mixed polymers. In certain embodiments, polynucleotide refers to polyribonucleotides, polydeoxynucleotides, or modified forms of any type of nucleotide, and combinations thereof. This term also includes, but is not limited to, single-stranded and double-stranded forms of DNA. In addition, polynucleotides, such as cDNA or mRNA, can contain either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-natural nucleotide bonds. Nucleic acid molecules can be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelators, alkylators, and modified linkages (e.g., alpha-anomeric nucleic acids, etc.). The above terms are also intended to encompass any topological structure, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpin, circular, and padlock structures. Unless otherwise specified, a reference to a nucleic acid sequence encompasses its complement. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, along with its complementary sequence. The term also includes codon-optimized nucleic acids encoding the same polypeptide sequence.

[0048] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0049] An "isolated nucleic acid encoding an antibody or fragment thereof" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and including such nucleic acid molecule(s) present in one or more locations in a host cell.

[0050] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which it is introduced. As used herein, "vector" refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0051] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. A host cell can be a recombinant host cell, and includes the primary transformed cell and progeny derived therefrom without regard to the number of passages.

[0052] A polypeptide "variant," as the term is used herein, is typically a polypeptide that differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. In the present invention, "variants" with reference to the sequences described in the "Anti-CSP Antibody Variants" section refer to engineered sequences rather than naturally occurring sequences.

[0053] As used herein, a "recombinant antibody" refers to an antibody whose exact amino acid sequence is not naturally found in a given organism (e.g., an antibody derived from a mammal). In certain embodiments, the term can refer to an antibody that contains one or more amino acid residues not found in a naturally occurring antibody. In certain embodiments, a recombinant antibody can have a CDR that contains amino acid residues not found in a naturally occurring antibody (e.g., an antibody derived from a mammal). In another exemplary embodiment, a recombinant antibody can have a framework (FR) that contains amino acid residues not found in a naturally occurring antibody (e.g., an antibody derived from a mammal). In certain embodiments, a recombinant antibody can have a constant region that contains amino acid residues not found in a naturally occurring antibody (e.g., an antibody derived from a mammal).

[0054] The term "equivalent" in the context of describing the binding strength of two antibodies to the same target refers to two dissociation constant (KD) values ​​calculated from two binding reactions that are within 3-fold of each other. In certain embodiments, the ratio of the first KD (KD of the binding reaction between the first antibody and the target) to the second KD (KD of the binding reaction between the second antibody and the target) is within an endpoint exclusive range of 1:3 or 3:1. A lower KD value indicates stronger binding. For example, but not limited to, an antibody variant with stronger binding compared to AB-000224 binds to the target with a KD that is at least one-third of the KD measured for AB-000224 against the same target.

[0055] Anti-CSP antibody The present disclosure provides anti-CSP antibodies and variants thereof. The malaria antibodies disclosed herein were discovered in antibody repertoires generated by Immune Repertoire Capture® (IRC®) technology from plasmablast B cells isolated from two donors enrolled in a Phase 2a study evaluating the efficacy of the RTS,S vaccine in preventing malaria infection. IRC® technology and its use in antibody discovery are well known and are disclosed, for example, in WO2012148497A2, the entire contents of which are incorporated herein by reference. The RTS,S vaccine is a pseudovirion vaccine that combines the central repeat and C-terminal region of the hepatitis B surface antigen and the Plasmodium falciparum (P. falciparum) circumsporozoite protein (CSP). RTS,S consists of two polypeptides: RTS is a single polypeptide chain corresponding to amino acids 207-395 of P. falciparum (3D7) fused to HBsAg, and S is a 226-amino acid polypeptide corresponding to HBsAg. (Stoute, et al., N Engl J Med;336:86-91(1997); RTS,S Clinical Trials Partnership, PLoS Med.11(7):e1001685,(2014), WO1993 / 10152).

[0056] CSPs contain three major domains: i) the N-terminus, ii) a central repeat (CR) region consisting of multiple (25–40) tetrapeptides of NANP ("major repeats") interspersed with NPDP and 2–4 NVDP ("minor repeats") tetrapeptides, and iii) a C-terminal domain. The central repeat region of CSPs is highly immunogenic and is present in all P. falciparum strains for which CSP sequences are available. The repeat region is composed of one NPDP repeat, three to five NVDP repeats, and 35–41 NANP repeats (e.g., P. falciparum strain 3D7 contains a total of 1 / 4 / 38 NPDP / NVDP / NANP motifs). The repeat region begins with a junctional NPDP sequence, typically followed by three alternating NANP and NVDP sequences, followed by the remaining NANP repeats. Most P. falciparum strains have one NVDP repeat interspersed in the middle of the long NANP repeat region. Pholcharee, T. et al., J.Mol.Bio.432:1048-1063(2020).

[0057] Analysis of anti-CSP antibodies disclosed herein demonstrated an inverse correlation between the percentage of CSP-specific IgG-expressing plasmablasts and protection in antibodies isolated after the third dose of vaccine (P3D). These data suggest that, despite the well-documented association between anti-CSP antibodies and protection, increasing the number of cells expressing anti-CSP, NANP repeat-binding antibodies may not confer greater protection. This result may indicate that the simple presence of potent inhibitory antibodies by P3D plasmablasts is insufficient for protection. Rather, the relative levels of such antibodies versus other repeat-binding antibodies may be important in providing consistent protection.

[0058] Our analysis further demonstrated that in vivo antibody sporozoite-inhibitory activity did not correlate with binding kinetics to the long NANP6 peptide (Table 9), but significantly correlated with koff for CSP and with binding kinetics to both the short NANP-containing peptide (NPNA3) and the minor repeat and junction region (JR) peptide (Table 9). These data suggest that the affinity of protective antibodies induced after RTS,S vaccination matures for short NANP repeats but also acquires or retains promiscuous binding activity for minor repeat and junction region epitopes not present in RTS,S. Consistent with this interpretation, the level of SHM aggregation in both the heavy and light chains of protective antibodies correlated with binding kinetics to short NANP-, NVDP-, and NPDP-containing peptides (Figures 1C–1E, Table 9) and inhibitory activity in a sporozoite challenge model (Figures 2H–2I, Table 9). Thus, affinity maturation and function appear to be related to short peptide sequences, both included and excluded from RTS,S, but not to long NANP repeat sequences.

[0059] Furthermore, the correlation between in vivo activity and binding kinetics to NVDP- and NPDP-containing peptides not present in RTS,S (Figure 2F-G, Table 9), the correlation between in vivo activity and binding kinetics to the short NPNA3 but not the long NANP6 peptide (Figure 2E, Table 9), and the inverse correlation between NANP6 reactivity of expanded antibody lines and protection against CHMI are consistent with the hypothesis that multiple NANP repeats act as immune "decoys" that deflect and dilute protective immunity. Under this hypothesis, antibody lines that bind only to NANP repeat region epitopes but provide limited protection are preferentially expanded, diluting the protective capacity of the broader anti-CSP repertoire. Promiscuous antibodies that also bind multiple NANP repeats and are similarly densely present on CSPs could further enhance antibody on-kinetics to heterologous epitopes. Therefore, the avidity provided by promiscuous binding could potentially promote a more protective response in vivo.

[0060] In certain embodiments, the anti-CSP antibodies disclosed herein bind to a first epitope present in the central repeat region of CSP and to a second epitope of CSP. In certain embodiments, the central repeat region of the CSP epitope comprises the amino acid sequence NPNA. Epitopes comprising NPNA include, for example, NPNANP, NANPNA, ANPNAN, NANPNANP, ANPNANPN, NPNANPNA, PNANPNAN, (NPNA)3, or (NPNA)4.

[0061] In certain embodiments, the anti-CSP antibodies disclosed herein bind to a second epitope, heterologous to the epitope present in the RTS,S vaccine, in addition to the epitope comprising NPNA, referred to herein as a heterologous epitope. Heterologous epitopes include epitopes in the minor repeat region of CSP, including the epitope comprising DPNA / NPNV, and epitopes in the junction region of CSP, including the epitope comprising DPNA.

[0062] Anti-CSP antibodies and their variants In certain embodiments, the present disclosure provides anti-CSP antibody variants of antibodies isolated from human subjects, in certain embodiments, the variants exhibit protective effects in vivo, as demonstrated, for example, by reduced parasite load in a mouse model of malaria infection.

[0063] In certain embodiments, the anti-CSP variants disclosed herein maintain the binding specificity, activity, and stability, and / or manufacturing characteristics of the parent antibody. In certain embodiments, the anti-CSP variants disclosed herein produced have improved developability, as determined, for example, through various in vitro assays such as aggregation assessment by HPLC or UPLC, hydrophobic interaction chromatography (HIC), multispecific assays (e.g., baculovirus particle binding), self-interacting nanoparticle spectroscopy (SINS), or mass spectrometry after incubation under accelerated degradation conditions such as high temperature, low pH, high pH, ​​or oxidized HO. A mutation is successful if activity is maintained (or enhanced) while eliminating or reducing the severity of a disadvantage.

[0064] The disadvantages of antibodies are further described in Table 1 below: [Table 1]

[0065] Another goal of engineering variants is to reduce the risk of clinical immunogenicity, for example, to reduce the generation of anti-drug antibodies against a therapeutic antibody. In certain embodiments, anti-CSP antibody variants have reduced immunogenicity compared to the parent antibody.

[0066] Factors that contribute to clinical immunogenicity can be divided into two groups: first, factors inherent to the drug, such as sequence, post-translational modifications, aggregates, degradation products, and contaminants; and second, factors related to the method of drug administration, such as dose level, dose frequency, route of administration, patient immune status, and patient HLA type.

[0067] One approach to engineering a variant to resemble itself as closely as possible is to identify a nearby germline sequence and mutate as many mismatched positions as possible (also known as "germline deviations") to the germline residue type. This approach applies to the germline genes IGHV, IGHJ, IGKV, IGKJ, IGLV, and IGLJ, which account for all of the variable heavy (V11) and variable light (VL) regions except for part of the H-CDR3. The germline gene IGHD encodes part of the H-CDR3 region, but typically exhibits too much variation in how it recombines with IGHV and IGHJ (e.g., forward or reverse, in any of the three translation frames, and in 5' and 3' modifications and non-templated additions) to represent a "self" sequence template from a population perspective.

[0068] Each germline gene may appear as different alleles in a population. In terms of minimizing the percentage of patients with immunogenic responses, the least immunogenic drug candidate is likely to be one that matches the alleles commonly found in the patient population. Single nucleotide polymorphism (SNP) data from the human genome can be used to approximate the frequency of alleles in a population.

[0069] Another approach to engineer leads to reduce immunogenicity risk is to use in silico predictions of immunogenicity, such as prediction of T cell epitopes, or in vitro assays of immunogenicity, such as ex vivo human T cell activation. For example, services such as those provided by Lonza, United Kingdom, are available that use platforms for HLA binding prediction and in vitro evaluation to further identify potential epitopes.

[0070] In certain embodiments, antibody variants are additionally designed to enhance the efficacy of the antibody.The design parameters of this embodiment focus on CDR, for example, CDR3.The position to be mutated is determined based on the structural analysis of antibody-antigen cocrystals (Oyen et al., Proc. Natl. Acad Sci. USA 114: E10438-E10445, 2017) and the sequence information of other antibodies from the same lineage as AB-000224 or AB-007088.

[0071] Approaches to Mutation Design The development disadvantages can be eliminated or reduced by one or more mutations. The mutations are designed to eliminate or reduce the development disadvantages and improve function while maintaining antibody structure and function. In certain embodiments, mutations to chemically similar residues are identified to maintain size, shape, charge, and / or polarity. Non-limiting examples of mutations are listed in Table 2 below: [Table 2]

[0072] In certain embodiments, variants of anti-CSP antibodies disclosed herein contain modifications relative to the parent antibody that provide improved pharmacokinetic properties, increased serum stability, stronger binding, and / or improved in vivo protective efficacy compared to the parent. In certain embodiments, variants of anti-CSP antibodies disclosed herein exhibit reduced immunogenicity and / or increased manufacturability compared to the parent. In certain embodiments, variants of anti-CSP antibodies disclosed herein have at least one modification, e.g., substitution, relative to the parent variable heavy or light chain sequences described herein, resulting in improved developability, e.g., reduced heterogeneity, increased yield, increased stability, improved net charge for improved pharmacokinetics, and / or reduced immunogenicity. In certain embodiments, the VH or VL regions of such variants of anti-CSP antibodies disclosed herein have at least two, three, four, five, six, or more modifications, e.g., substitutions.

[0073] In certain embodiments, variants of anti-CSP antibodies disclosed herein exhibit increased serum half-life compared to the parent antibody. In certain embodiments, variants of anti-CSP antibodies disclosed herein have at least one modification, e.g., substitution, relative to the native Fc region of the heavy or light chain sequences described herein, and have improved pharmacokinetic properties, e.g., half-life. In certain embodiments, the heavy chain Fc region or light chain Fc region of such variants of anti-CSP antibodies disclosed herein have at least two, three, four, five, six, or more modifications, e.g., substitutions. In certain embodiments, variants of anti-CSP antibodies disclosed herein have a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modifications, e.g., substitutions, including both the heavy and light chains, compared to the parent antibody. In certain non-limiting embodiments, the Fc region of the heavy chain of a variant of an anti-CSP antibody disclosed herein can comprise an isoleucine at position 250, a tyrosine at position 252, an isoleucine at position 259, a glutamine at position 307, a phenylalanine at position 308, a leucine at position 319, a leucine at position 428, a histidine at position 434, a phenylalanine at position 434, an alanine at position 434, a serine at position 434, a methionine at position 434, or a combination thereof, where the numbering is defined by the EU index in Kabat. In certain embodiments, the Fc region of the heavy chain of a variant of an anti-CSP antibody disclosed herein comprises a leucine at position 428 and a serine at position 434, where the numbering is defined by the EU index in Kabat.

[0074] Antibody sequences and variants thereof In certain embodiments, the anti-CSP antibodies disclosed herein comprise a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NOs: 1-461 in Table 3. In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NOs: 462-922 in Table 3. In certain embodiments, a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NOs: 1-461, and b) the light chain variable region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NOs: 462-922.

[0075] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NOs: 1 to 461 shown in Table 3. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NOs: 462 to 922 shown in Table 3. In certain embodiments, a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NOs: 1 to 461, and b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NOs: 462 to 922.

[0076] In certain embodiments, the heavy chain variable region consists of the amino acid sequence set forth in SEQ ID NOs: 1 to 461 in Table 3. In certain embodiments, the light chain variable region consists of the amino acid sequence set forth in SEQ ID NOs: 462 to 922 in Table 3. In certain embodiments, a) the heavy chain variable region consists of the amino acid sequence set forth in SEQ ID NOs: 1 to 461, and b) the light chain variable region consists of the amino acid sequence set forth in SEQ ID NOs: 462 to 922.

[0077] In certain embodiments, the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the heavy chain variable sequences set forth in SEQ ID NOs: 1 to 461 shown in Table 3. In certain embodiments, the light chain variable region comprises CDR1, CDR2, and CDR3 of the light chain variable sequences set forth in SEQ ID NOs: 462 to 922 shown in Table 3. In certain embodiments, a) the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the heavy chain variable sequences set forth in SEQ ID NOs: 1 to 461, and b) the light chain variable region comprises CDR1, CDR2, and CDR3 of the light chain variable sequences set forth in SEQ ID NOs: 462 to 922.

[0078] In certain embodiments, anti-CSP antibodies disclosed herein comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 and having the amino acid sequences set forth in SEQ ID NOS: 1-461 as shown in Table 3. In certain embodiments, anti-CSP antibody variants comprise a light chain variable region comprising CDR1, CDR2, and CDR3 and having the amino acid sequences set forth in SEQ ID NOS: 462-922 as shown in Table 3. In certain embodiments, anti-CSP antibody variants comprise a) a heavy chain variable region comprising CDR1, CDR2, and CDR3 and having the amino acid sequences set forth in SEQ ID NOS: 1-461, and b) a light chain variable region comprising CDR1, CDR2, and CDR3 and having the amino acid sequences set forth in SEQ ID NOS: 462-922. Table 3 is provided below.

[0079] In certain embodiments, anti-CSP antibody variants are designated as shown in Table 3. For example, without limitation, an anti-CSP antibody variant comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 462 is designated as "AB-001558." [Table 3-1] [Table 3-2] [Table 3-3] Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21 Table 3-22 Table 3-23 Table 3-24 Table 3-25 Table 3-26 Table 3-27 Table 3-28 Table 3-29 Table 3-30 Table 3-31 Table 3-32 Table 3-33 Table 3-34 Table 3-35 Table 3-36 Table 3-37 Table 3-38 Table 3-39 Table 3-40 Table 3-41 Table 3-42 Table 3-43 Table 3-44 Table 3-45 Table 3-46 Table 3-47 Table 3-48 Table 3-49 Table 3-50 Table 3-51 Table 3-52 Table 3-53 Table 3-54 [Table 3-55] [Table 3-56] [Table 3-57] [Table 3-58] [Table 3-59] [Table 3-60] [Table 3-61] [Table 3-62] [Table 3-63] [Table 3-64] [Table 3-65] [Table 3-66]

[0080] epitope In certain embodiments, the anti-CSP antibodies disclosed herein bind to the first epitope of CSP. CSP consists of an N-terminal domain containing a heparan sulfate binding site for hepatocyte adhesion, a central repeat region, and structured C-terminal α-thrombospondin repeats (αTSR), followed by a GPI anchor that connects CSP to the sporozoite membrane. The central repeat region of CSP is highly immunogenic and is present in all P. falciparum strains for which CSP sequences are available. The repeat region is composed of one NPDP repeat, three to five NVDP repeats, and 35 to 41 NANP repeats (e.g., P. falciparum strain 3D7 contains a total of 1 / 4 / 38 NPDP / NVDP / NANP motifs). The repeat region begins with a junction NPDP sequence, typically followed by three alternating NANP and NVDP sequences, followed by the remaining NANP repeats; most P. falciparum strains have one NVDP repeat interspersed in the middle of the long NANP repeat region. Pholcharee, T. et al., J.Mol.Bio.132:1048-1063(2020).

[0081] In certain embodiments, the anti-CSP antibodies disclosed herein bind to the central repeat region of P. falciparum CSP. In certain embodiments, the antibodies disclosed herein bind to the P. falciparum CSP protein at the repeat and / or junction region containing the NPNA, NPDP, and / or NVDP motifs. In certain embodiments, the anti-CSP antibodies disclosed herein bind to the NANP repeat region of P. falciparum CSP. In certain embodiments, the anti-CSP antibodies disclosed herein bind to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:923.

[0082] In certain embodiments, the first epitope comprises the amino acid sequence set forth in SEQ ID NOs: 923 to 974. In certain embodiments, the first epitope consists of the amino acid sequence set forth in SEQ ID NOs: 923 to 974. SEQ ID NOs: 923 to 974 are provided in Table 4 below. [Table 4]

[0083] In certain embodiments, the anti-CSP antibodies disclosed herein further bind to a second epitope of CSP. In certain embodiments, the second epitope is heterologous to the epitope present in the RTS,S vaccine. The RTS,S vaccine is a pseudovirion vaccine that combines the central repeat and C-terminal regions of the hepatitis B surface antigen and the Plasmodium falciparum (P. falciparum) circumsporozoite protein (CSP). RTS,S consists of two polypeptides: RTS, a single polypeptide chain corresponding to amino acids 207-395 of P. falciparum (3D7) fused to HBsAg, and S, a 226-amino acid polypeptide corresponding to HBsAg. Stoute,et.al.,N Engl J Med;336:86-91(1997);RTS,S Clinical Trials Partnership,PLoS Med.11(7):e1001685,(2014), WO1993 / 10152.

[0084] In certain embodiments, the second epitope comprises a minor repeat or junction region of a CSP. In certain embodiments, the second epitope comprises a minor repeat and junction region of a CSP. In certain embodiments, the minor repeat is a DPNA / NPNV-containing minor repeat. In certain embodiments, the junction region is a DPNA / NPNV-containing junction region. In certain embodiments, the second epitope comprises the amino acid sequence set forth in SEQ ID NOs: 975-1195. In certain embodiments, the second epitope consists of the amino acid sequence set forth in SEQ ID NOs: 975-1195. SEQ ID NOs: 975-1195 are provided in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3]

[0085] In certain embodiments, the anti-CSP antibodies disclosed herein specifically bind to a first epitope of CSP and a second epitope of CSP. In certain embodiments, the anti-CSP antibodies bind to at least one additional epitope of CSP. In certain embodiments, the first epitope comprises the amino acid sequence set forth in SEQ ID NOs: 923-974, and the second epitope comprises the amino acid sequence set forth in SEQ ID NOs: 975-1195. In certain embodiments, the first epitope consists of the amino acid sequence set forth in SEQ ID NOs: 923-974, and the second epitope consists of the amino acid sequence set forth in SEQ ID NOs: 975-1195. In certain embodiments, the first epitope consists of the amino acid sequence set forth in SEQ ID NOs: 923-974, the second epitope consists of the amino acid sequence set forth in SEQ ID NOs: 975-1195, and at least one additional epitope consists of the amino acid sequence set forth in SEQ ID NOs: 975-1195.

[0086] Glycosylation of anti-CSP antibodies and their variants Glycosylation of antibodies and engineered antibodies has been previously disclosed (see, e.g., U.S. Patent No. 6,602,684, the contents of which are incorporated in their entirety). Antibody Fc regions are generally post-translationally modified through the addition of N-glycans at specific asparagine residues on the antibody heavy chain. IgG molecules have an N-linked glycosylated asparagine on each heavy chain. It has been shown that modified glycosylation profiles can modulate antibody function. For example, but not limited to, altered glycosylation can improve the binding affinity or half-life of an antibody compared to the unmodified form.

[0087] In certain embodiments, the present disclosure provides anti-CSP antibodies and variants thereof with modified glycosylation. In certain embodiments, the antibodies disclosed herein comprise an Fc region with increased glycosylation. In certain non-limiting embodiments, the Fc region with increased glycosylation comprises an increased amount of bisected oligosaccharides. In certain embodiments, the Fc region with increased glycosylation comprises an increased amount of nonfucosylated oligosaccharides. In certain embodiments, the Fc region with increased glycosylation comprises an increased amount of fucose-containing oligosaccharides.

[0088] In certain embodiments, the antibodies disclosed herein comprise an Fc region with reduced glycosylation. In certain non-limiting embodiments, an Fc region with reduced glycosylation comprises a reduced amount of bisected oligosaccharides. In certain embodiments, an Fc region with reduced glycosylation comprises a reduced amount of nonfucosylated oligosaccharides. In certain embodiments, an Fc region with increased glycosylation comprises a reduced amount of fucose-containing oligosaccharides.

[0089] In certain embodiments, the antibodies disclosed herein comprise V regions with increased glycosylation. In certain non-limiting embodiments, the V regions with increased glycosylation comprise increased amounts of bisected oligosaccharides. In certain embodiments, the V regions with increased glycosylation comprise increased amounts of nonfucosylated oligosaccharides. In certain embodiments, the V regions with increased glycosylation comprise increased amounts of fucose-containing oligosaccharides.

[0090] In certain embodiments, the antibodies disclosed herein comprise V regions with reduced glycosylation. In certain non-limiting embodiments, V regions with reduced glycosylation comprise reduced amounts of bisected oligosaccharides. In certain embodiments, V regions with reduced glycosylation comprise reduced amounts of nonfucosylated oligosaccharides. In certain embodiments, V regions with increased glycosylation comprise reduced amounts of fucose-containing oligosaccharides.

[0091] In certain embodiments, modified glycosylation can be obtained by expressing any of the antibodies disclosed herein in a host cell with altered glycosylation machinery. For example, but not limited to, the host cell can contain a functional disruption of a fucosyltransferase gene, such that antibodies expressed in the host cell exhibit reduced glycosylation, e.g., reduced fucosylation (see PCT Patent Publication No. WO 99 / 54342).

[0092] In certain embodiments, the present disclosure provides anti-CSP antibody variants disclosed herein that include one or more amino acid substitutions that result in an alteration of a glycosylation acceptor site. In certain embodiments, the alteration includes removal of a glycosylation acceptor site. In certain embodiments, the alteration includes modification of a glycosylation acceptor site. In certain embodiments, the alteration includes insertion of a glycosylation acceptor site.

[0093] As used herein, "glycosylation acceptor site" refers to an amino acid residue in the light or heavy chain of an antibody that can be N- or O-glycosylated. In certain embodiments, an N-linked glycosylation acceptor site can be an asparagine residue. In certain embodiments, an O-linked glycosylation acceptor site can be a serine, threonine, tyrosine, hydroxylysine, or hydroxyproline residue.

[0094] In certain embodiments, the Fc region of an antibody disclosed herein comprises one or more glycosylation acceptor sites. In certain embodiments, the V region of any of the antibodies disclosed herein comprises one or more glycosylation acceptor sites. In certain embodiments, the light chain of any of the antibodies disclosed herein comprises one or more glycosylation acceptor sites. In certain embodiments, the heavy chain of any of the antibodies disclosed herein comprises one or more glycosylation acceptor sites. In certain embodiments, the light chain variable region of any of the antibodies disclosed herein comprises one or more glycosylation acceptor sites. In certain embodiments, the heavy chain variable region of any of the antibodies disclosed herein comprises one or more glycosylation acceptor sites.

[0095] PEGylation and other chemical modifications of anti-CSP antibodies and their variants The present disclosure provides anti-CSP antibodies and variants thereof containing additional modifications. In certain embodiments, the modifications can improve the pharmacological properties of the antibody, such as half-life. In certain non-limiting embodiments, the modifications include PEGylation, deamination, derivatization with a polymer, lipidation, removal and / or introduction of disulfide bonds, oxidation, and removal of the C-terminal lysine.

[0096] In certain embodiments, the modification is PEGylation. PEGylation of antibodies and engineered antibodies involves the attachment of one or more polyethylene glycols (PEGs) to the antibody. In certain non-limiting embodiments, for example, PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to any of the forms of PEG that have been used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide.

[0097] In certain embodiments, the modification is derivatization with a hydrophilic polymer. In certain non-limiting embodiments, for example, the hydrophilic polymer can be carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0098] In certain embodiments, the modification is lipidation. Lipidation is the conjugation of proteins with lipids. The lipidation of peptides improves metabolic stability, membrane permeability, bioavailability, and changes the pharmacokinetic and pharmacodynamic properties of peptides. For example, lipidated peptides have a high affinity with serum albumin, which leads to increased half-life and stability. In certain non-limiting embodiments, for example, the lipid can be myristic acid, palmitic acid, stearic acid, lauric acid, cholesterol, and mixtures thereof.

[0099] In certain embodiments, the modification is a substitution of an amino acid residue to form a disulfide bond. In certain embodiments, the amino acid substitution introduces a cysteine. Under certain redox conditions, two cysteines may form a non-native disulfide bond. In certain non-limiting embodiments, the disulfide bond improves antibody stability, for example, correcting antibody chain pairing. In certain embodiments, a cysteine ​​is introduced into the V region. In certain embodiments, a cysteine ​​is introduced into the Fc region. In certain embodiments, the modification is a substitution of an amino acid residue to eliminate a disulfide bond. In certain embodiments, the amino acid substitution removes a cysteine. In certain embodiments, the cysteine ​​is substituted with serine. In certain non-limiting embodiments, removing the cysteine ​​improves antibody stability, for example, improving long-term stability. In certain embodiments, the cysteine ​​is removed in the V region. In certain embodiments, the cysteine ​​is removed in the Fc region.

[0100] Anti-CSP antibodies and anti-CSP antibody variant conjugates In certain embodiments, the present disclosure provides an anti-CSP antibody or variant thereof conjugated or bound to a therapeutic and / or imaging / detectable moiety. For example, but not limited to, the anti-CSP antibody or variant thereof can be conjugated to a detectable marker, a toxin, or a therapeutic agent. The moiety can be covalently or non-covalently bound to the antibody.

[0101] In certain embodiments, the antibody or variant thereof is conjugated to a cytotoxic moiety or other moiety that inhibits cell proliferation. In certain embodiments, the antibody or variant thereof is conjugated to any of the following: ricin A chain, doxorubicin, daunorubicin, maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin A, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alphasarcin, gelonin, mitogellin, restrictocin, kobran venom factor, ribonuclease, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitors, glucocorticoids, auristatins, auromycins, yttrium, bismuth, combrestatins, duocarmycins, dolastatins, cc1065, or cisplatin. In certain embodiments, the antibody or variant thereof can be conjugated to a drug such as an enzyme inhibitor, a growth inhibitor, a lytic agent, a DNA or RNA synthesis inhibitor, a membrane permeability modifier, a DNA metabolite, a dichloroethyl sulfide derivative, a protein production inhibitor, a ribosome inhibitor, or an inducer of apoptosis.

[0102] In certain embodiments, the antibody or variant thereof can be conjugated to a radionuclide, iron-related compound, dye, fluorescent agent, or imaging agent. In certain embodiments, the antibody can be conjugated to an agent such as, but not limited to, a metal, a metal chelator, a lanthanide, a lanthanide chelator, a radiometal, a radiometal chelator, a positron-emitting nucleus, a microbubble (for ultrasound), a liposome, a molecule microencapsulated in a liposome or nanosphere, a single-crystalline iron oxide nanocompound, a magnetic resonance imaging contrast agent, a light absorbing, reflecting, and / or scattering agent, a colloidal particle, a fluorophore such as a near-infrared fluorophore, etc.

[0103] In certain embodiments, the present disclosure provides bispecific molecules comprising the anti-CSP antibodies, variants thereof, or fragments thereof disclosed herein. The anti-CSP antibodies, anti-CSP antibody variants, or antigen-binding portions thereof can be derivatized or conjugated to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to generate bispecific molecules that bind to at least two different binding sites or target molecules. The anti-CSP antibodies or variants thereof disclosed herein can be derivatized or conjugated to two or more other functional molecules to generate multispecific molecules that bind to three or more different binding sites (e.g., two different epitopes on a CSP protein) and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create the bispecific molecules of the present invention, the antibodies of the present invention can be functionally conjugated (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, to generate bispecific molecules. In certain non-limiting embodiments, for example, but not limited to, bispecific antibodies can be created using a knobs-in-holes strategy. This strategy typically involves creating a first half of a first antibody that recognizes a first antigen, e.g., CSP, and a second half of an antibody that recognizes a second antigen or binding site, and then combining the two halves to create the bispecific antibody. In certain embodiments, the first antigen and the second antigen are different epitopes of the CSP protein.

[0104] activity The activity of any of the anti-CSP antibodies disclosed herein can be evaluated by using different endpoints. In certain embodiments, activity is evaluated for binding to CSP, either by binding to a series of linear peptides of various lengths representing the immunodominant region of the CSP protein, or by binding to the entire CSP protein. In certain embodiments, activity is evaluated for the ability to protect against challenge with Plasmodium containing P. falciparum CSP, for example, in an in vivo animal model of malaria. In certain embodiments, effector function, such as ADCC, is also evaluated.

[0105] In certain embodiments, the binding activity of the anti-CSP antibodies disclosed herein to P. falciparum CSP protein can be assessed by surface plasmon resonance (SPR) using a biosensor system. Suitable systems for use with SPR include, but are not limited to, the LSA™ (Carterra, Dublin, CA), Biacore™ (General Electric, Boston, MA), and OpenSPR (Nicoya, East Kitchener, ON, Canada). In an exemplary SPR assay, each antibody can be directly immobilized on a Carterra CMD200M chip or captured on a CMD200M Carterra chip using a goat anti-human IgG Fc antibody. Unbound antibody can be washed away, and a gradient of target concentrations can be applied over the antibody. In certain experimental conditions, the highest concentration of each target can range from 0.5 to 8 μg / mL. For better precision, each antibody can be immobilized at different locations (e.g., at least two) on the chip, and the affinity for each antibody-target combination can be determined using multiple (e.g., four to five) target concentrations according to standard methods. If the variation between two replicates exceeds three-fold, repeat the antibody-target measurements.

[0106] In certain embodiments, the binding activity of anti-CSP antibodies disclosed herein to P. falciparum CSP proteins can be assessed by biolayer interferometry (BLI). For BLI, each antigen can be immobilized on a sensor according to the manufacturer's instructions. Suitable systems for use in BLI include, but are not limited to, Octet™ (ForteBio, Fremont, CA) and Gator™ (Probelife, Palo Alto, CA). In certain embodiments, for example and without limitation, the antigen can be biotinylated and immobilized on a streptavidin sensor. For better precision, each antibody can be assessed in duplicate at a suitable concentration (e.g., 5 μg / mL). If the variation between two duplicates exceeds three-fold, the antibody-target measurement is repeated. Assays are typically performed under conditions according to the manufacturer's instructions. The assay can be performed at a temperature ranging from 20°C to 37°C, e.g., from 20°C to 25°C. In certain embodiments, the assay is performed at 25°C. In certain embodiments, the assay is performed at 37°C.

[0107] In certain embodiments, binding to the CSP protein is assessed in a competition assay format with reference antibody A. In certain embodiments, the variant anti-CSP antibodies disclosed herein are capable of blocking binding of the reference antibody by about 50% or more in the competition assay.

[0108] The anti-CSP antibodies and anti-CSP antibody variants of the present disclosure can also be evaluated in various assays for their ability to mediate FcR-dependent activities.

[0109] In certain embodiments, the activity of anti-CSP antibodies can be evaluated in vivo in animal models, for example, as described in the Examples section. In certain non-limiting embodiments, a mouse malaria liver burden assay can be used, for example, as disclosed in Flores-Garcia Y, et al. Malar J. 2019;18(1):426, doi:10.1186 / s12936-019-3055-9, the contents of which are incorporated herein by reference. Mice are administered the antibody and infected with transgenic P. berghei expressing GFP-luciferase and P. falciparum CSP protein. Parasite liver burden can be assessed, for example, by RT-qPCR or by measuring bioluminescence using an IVIS spectrum imaging device. A reduction in parasite liver burden reflects the preventive activity of the antibody.

[0110] In certain embodiments, the activity of anti-CSP antibodies can be determined by assessing in vivo protection and survival in an animal model, such as mice. For example, but not limited to, mice are administered with the antibody and challenged with transgenic P. berghei expressing P. falciparum CSP protein. In vivo protection can be determined by detecting blood-stage parasitemia under a microscope. Survival can be determined using the absence of parasitemia during an observation period immediately after challenge, for example, two weeks. Increased survival reflects the preventive and / or therapeutic activity of the antibody.

[0111] In certain embodiments, the anti-CSP antibodies disclosed herein have at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or 70%, or more of the activity of antibody AB-000317 when evaluated under the same assay conditions. In certain embodiments, the anti-CSP antibodies exhibit improved activity compared to antibody AB-000317, i.e., greater than 100% activity. In certain non-limiting embodiments, the anti-CSP antibodies disclosed herein exhibit at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or 70%, or more reduction in parasite liver burden compared to antibody AB-000317. In certain non-limiting embodiments, the anti-CSP antibodies disclosed herein exhibit an increase in survival rate of at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or 70%, or more, compared to antibody AB-000317.

[0112] In certain embodiments, the anti-CSP antibody variants disclosed herein have at least 50%, or at least 60%, or 70%, or more of the activity of AB-000224 when evaluated under the same assay conditions. In certain embodiments, the anti-CSP antibodies exhibit improved activity compared to AB-000224, i.e., greater than 100% activity. In certain embodiments, the anti-CSP antibody variants disclosed herein have similar activity against malaria infection compared to AB-000224. In certain embodiments, the anti-CSP antibody variants disclosed herein have at least 50%, or at least 60%, or 70%, or more of the activity of AB-007088 when evaluated under the same assay conditions. In certain embodiments, the anti-CSP antibodies exhibit improved activity compared to AB-007088, i.e., greater than 100% activity. In certain embodiments, the anti-CSP antibody variants disclosed herein have similar activity against malaria infection compared to AB-007088. The term "similar activity," when used to compare the in vivo activity of antibodies, means that two measurements of activity differ from each other by no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 8%, or no more than 5%.

[0113] Antibody generation The CSP antibodies and variants thereof disclosed herein can be produced using vectors and recombinant methodologies (see, e.g., Sambrook & Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Ausubel, Current Protocols in Molecular Biology). Reagents, cloning vectors, and kits for genetic manipulation are available from commercial suppliers.

[0114] The present disclosure provides isolated nucleic acids encoding the VH and / or VL regions, or fragments thereof, of any of the anti-CSP antibodies and anti-CSP antibody variants disclosed herein. In certain embodiments, the present disclosure provides vectors containing the antibody-encoding nucleic acids, used to replicate the nucleic acids and / or to express the antibodies, and host cells into which the nucleic acids are introduced. These nucleic acids can encode a VL-containing amino acid sequence and / or a VH-containing amino acid sequence (e.g., the light chain and / or heavy chain of the antibody) of an anti-CSP antibody or variant thereof. In certain embodiments, the host cell contains (1) a vector containing a polynucleotide encoding a VL amino acid sequence and a polynucleotide encoding a VH amino acid sequence, or (2) a first vector containing a polynucleotide encoding a VL amino acid sequence and a second vector containing a polynucleotide encoding a VH amino acid sequence.

[0115] In certain embodiments, the present disclosure provides a method for producing an anti-CSP antibody disclosed herein. In certain embodiments, the method comprises culturing the aforementioned host cells under conditions suitable for expression of the antibody. In certain embodiments, the antibody is then recovered from the host cells (or host cell culture medium).

[0116] Suitable vectors containing a polynucleotide encoding an antibody of the present disclosure, or a fragment thereof, include cloning vectors and expression vectors. While the cloning vector selected can vary according to the host cell intended for use, useful cloning vectors will generally be capable of autonomous replication, have a single target for a particular restriction endonuclease, and / or carry a gene for a marker that can be used to select clones containing the vector. Non-limiting examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColEl plasmids, pCR1, RP4, phage DNA, and shuttle vectors.

[0117] Expression vector is generally a replicable polynucleotide construct that contains the nucleic acid of the present disclosure.Expression vector can replicate in host cell either as episome or as an integral part of chromosomal DNA.Suitable expression vector includes but is not limited to plasmid and viral vector, including adenovirus, adeno-associated virus, retrovirus and any other vector.

[0118] Suitable host cells for expressing the anti-CSP antibodies or anti-CSP antibody variants disclosed herein include both prokaryotic and eukaryotic cells. For example, but not limited to, anti-CSP antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, the antibody can be isolated from bacterial cell lysates in a soluble fraction and further purified. Alternatively, the host cell can be a eukaryotic host cell, including, but not limited to, filamentous fungi or yeast, fungal and yeast strains in which the glycosylation pathway has been "humanized" to result in the production of antibodies with partially or fully human glycosylation patterns, vertebrate, invertebrate, and plant cells, and other eukaryotic microorganisms. Non-limiting examples of invertebrate cells include insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells. Plant cell cultures can also be used as host cells.

[0119] In certain embodiments, vertebrate host cells are used to produce the anti-CSP antibodies of the present disclosure. For example, mammalian cell lines that can be used to express anti-CSP antibodies include, but are not limited to, SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK, buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT060562), TRI cells, MRC5 cells, and FS4 cells. In certain embodiments, the mammalian cell line used to express anti-CSP antibodies is a Chinese hamster ovary (CHO) cell line, a DHFR-CHO cell line (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980), as well as myeloma cell lines such as YO, NSO, and Sp2 / 0. Host cells of the present disclosure also include, but are not limited to, isolated cells, in vitro cultured cells, and ex vivo cultured cells.

[0120] Host cells transfected with an expression vector encoding an anti-CSP antibody or fragment thereof of the present disclosure can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide can be secreted and isolated from the mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide can be retained in the cytoplasm or a membrane fraction, and the cells harvested, lysed, and the polypeptide isolated using a desired method.

[0121] Pharmaceutical compositions and methods of treatment In certain embodiments, the present disclosure provides pharmaceutical compositions for the administration of anti-CSP antibodies and variants thereof. In certain embodiments, the pharmaceutical compositions can be administered in a therapeutically effective amount to a mammalian subject, e.g., a human, having or at risk for malaria, according to a schedule sufficient to prevent Plasmodium infection, e.g., infection with Plasmodium falciparum or Plasmodium sp. having a cross-reactive CSP protein, or to alleviate symptoms of malaria in the subject. In certain embodiments, the pharmaceutical composition can comprise any of the anti-CSP antibodies and variants thereof disclosed herein, or a polynucleotide encoding same, and a pharmaceutically acceptable diluent or carrier. In certain embodiments, the polynucleotide encoding the antibody can be contained in a plasmid vector or a viral vector for delivery. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the antibody. As used herein, a "therapeutically effective dose" or "therapeutically effective amount" refers to an amount sufficient to prevent, cure, or at least partially prevent malaria or symptoms of malaria. A therapeutically effective dose can be determined by monitoring a patient's response to therapy. A typical benchmark for indicating a therapeutically effective dose is, for example, the improvement or prevention of malaria symptoms in a patient, including, but not limited to, a reduction in the number of parasites. The amount effective for this use will depend on the severity of the disease and the general state of the patient's health, including other factors such as age, weight, sex, and route of administration. Single or multiple administrations of the antibody will be administered as needed, depending on the dosage and frequency tolerated by the patient.

[0122] In certain embodiments, the antibody is administered at the pre-erythrocytic stage of infection, ie, the antibody is administered in a time frame to prevent or reduce hepatocyte infection.

[0123] Various pharmaceutically acceptable diluents, carriers, and excipients, and techniques for preparing and using pharmaceutical compositions are also disclosed herein.Exemplary pharmaceutical compositions and pharmaceutically acceptable diluents, carriers, and excipients are also described in Remington: The Science and Practice of Pharmacy 20th Ed. (Lippincott, Williams & Wilkins 2012).In certain embodiments, each carrier, diluent, or excipient is "acceptable" in the sense that it is compatible with the other components of the pharmaceutical composition and is not harmful to the subject.In many cases, the pharmaceutically acceptable carrier is a pH buffered aqueous solution. In certain non-limiting embodiments, for example, pharmaceutically acceptable carriers, diluents, or excipients include water; buffer solutions, e.g., phosphate-buffered saline; sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0124] In certain embodiments, the pharmaceutical composition can be formulated for any suitable route of administration, including, for example, parenteral, pulmonary, intranasal, or topical administration. Parenteral administration can include intramuscular, intravenous, intraarterial, intraperitoneal, oral, or subcutaneous administration. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration and has an antibody concentration of 10-100 mg / ml, 10-50 mg / ml, 20-40 mg / ml, or about 30 mg / ml. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous injection and has an antibody concentration of 50-500 mg / ml, 50-250 mg / ml, or 100-150 mg / ml and a viscosity of less than 50 cP, less than 30 cP, less than 20 cP, or less than about 10 cP. In certain embodiments, the pharmaceutical composition is liquid or solid. In certain embodiments, the pharmaceutical composition is formulated for parenteral administration, for example, intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

[0125] In certain embodiments, the formulation and delivery method of the pharmaceutical composition are adapted according to the site and disease to be treated. For example, but not limited to, formulations include formulations in which the antibody is encapsulated in micelles, liposomes, or drug-release capsules (active agent incorporated into a biocompatible coating designed for sustained release), ingestible formulations, topical formulations such as creams, ointments, and gels, and other formulations such as inhalants, aerosols, and sprays.

[0126] In certain non-limiting embodiments, for example, for parenteral administration, antibody or its antigen-binding fragment is formulated in unit dosage injectable form (solution, suspension, emulsion) in association with pharmaceutically acceptable parenteral vehicle.Non-limiting examples of vehicle include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.Non-aqueous vehicles such as fixed oil and ethyl oleate can also be used.

[0127] The dose and dosage regimen will depend on various factors, such as the nature of the infection, the subject's characteristics, and the subject's medical history, which can be easily determined by a physician. In certain embodiments, the amount of antibody or antigen-binding fragment thereof administered or provided to a subject is in the range of about 0.1 mg / kg to about 50 mg / kg of the subject's body weight. Depending on the type and severity of the infection, in certain embodiments, about 0.1 mg / kg to about 50 mg / kg of body weight (e.g., about 0.1 to 15 mg / kg / dose) of antibody or antigen-binding fragment thereof may be provided to a patient as an initial candidate dosage, for example, by one or more separate administrations or by continuous infusion. The progress of therapy is easily monitored by conventional methods and assays and based on criteria known to physicians or other skilled artisans.

[0128] The antibody or variant thereof of the present disclosure can be administered to a subject according to known methods using any route of administration, for example, systemically, parenterally, or locally. Such routes include, but are not limited to, intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or by inhalation routes. A subject can be administered one or more doses of the antibody of the present invention, which can be administered before, after, or simultaneously with another therapeutic agent, as further described below.

[0129] In certain embodiments, the antibodies or variants thereof of the present disclosure can be administered to prevent malaria. In certain embodiments, the antibodies disclosed herein can inhibit or reduce the risk of Plasmodium infection. In certain embodiments, the antibodies disclosed herein can inhibit or reduce the pre-erythrocytic or sporozoite stage of infection. In certain embodiments, the antibodies disclosed herein can prevent malaria by targeting Plasmodium at the early stage of invasion into the target vertebrate, thereby preventing infection from occurring.

[0130] In certain embodiments, the anti-CSP antibodies of the present disclosure can be administered to treat malaria. In certain embodiments, the antibodies disclosed herein can inhibit or reduce the progression of Plasmodium infection in the bloodstream. In certain embodiments, the antibodies disclosed herein can inhibit or reduce the risk of transmission of Plasmodium from one subject to another via insect feeding, such as mosquito bites, or contact with infected blood.

[0131] In certain embodiments, the pharmaceutical compositions disclosed herein can be administered to pediatric patients. As used herein, the term "pediatric patient" refers to patients up to 18 years of age. In certain embodiments, a pediatric patient is a patient between 3 months and 12 years of age. In certain non-limiting embodiments, a pediatric patient can be about 1 to about 2 years of age, about 2 to about 3 years of age, about 3 to about 4 years of age, about 4 to about 5 years of age, about 5 to about 6 years of age, about 6 to about 7 years of age, about 7 to about 8 years of age, about 8 to about 9 years of age, about 9 to about 10 years of age, or about 11 to about 12 years of age. In certain embodiments, the pediatric patient is unresponsive or poorly responsive to other treatments for malaria. In certain embodiments, the pediatric patient is a human.

[0132] In certain embodiments, the dose of the pharmaceutical composition disclosed herein is administered based on the body weight of the pediatric patient. In certain non-limiting embodiments, the dose of the pharmaceutical composition is about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, or about 350 mg / kg. In certain embodiments, the pediatric patient weighs about 2.5 kg to about 5 kg, about 5 kg to about 10 kg, about 10 kg to about 15 kg, about 15 kg to about 20 kg, about 20 kg to about 30 kg, or about 30 kg to about 40 kg.

[0133] In certain embodiments, the antibody is provided to a subject in combination with one or more additional therapeutic agents used to treat or prevent malaria or related diseases or disorders.In certain embodiments, a method for treating or preventing malaria is provided, comprising administering to a human a therapeutically effective amount of the antibody disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of one or more additional therapeutic agents.In certain embodiments, a method for treating malaria in a human who has infection or is at risk of infection is provided, comprising administering to a human a therapeutically effective amount of the antibody disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of one or more additional therapeutic agents.

[0134] In certain embodiments, when the antibodies of the present disclosure described herein are combined with one or more additional therapeutic agents as described above, the components of the composition are administered simultaneously or sequentially. When administered sequentially, the combination may be administered in two or more doses.

[0135] In certain embodiments, the antibodies disclosed herein are combined with one or more additional therapeutic agents in a single dosage form for simultaneous administration to a patient.

[0136] "Patient" refers to any subject receiving an antibody, whether or not they have malaria. In certain embodiments, a "patient" is a non-human subject, e.g., an animal used as a model to evaluate the effects of antibody administration.

[0137] "Co-administration" of an antibody disclosed herein with one or more additional therapeutic agents generally refers to simultaneous or sequential administration of an antibody disclosed herein, or fragment thereof, and one or more additional therapeutic agents such that therapeutically effective amounts of both the antibody disclosed herein, or fragment thereof, and the one or more additional therapeutic agents are present in the patient. Co-administration includes administration of a unit dosage of an antibody disclosed herein before or after administration of a unit dosage of the one or more additional therapeutic agents, for example, but not limited to, administration of the antibody within seconds, minutes, or hours of administration of the one or more additional therapeutic agents. In certain non-limiting embodiments, for example, a unit dose of an antibody disclosed herein is administered first, followed within seconds or minutes by a unit dose of one or more additional therapeutic agents. In certain non-limiting embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed within seconds or minutes by a unit dose of the antibody. In certain embodiments, a unit dose of an antibody disclosed herein is administered first, followed, after a period of time (e.g., 1-12 hours), by administration of a unit dose of one or more additional therapeutic agents. In certain embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed after a period of time (e.g., 1-12 hours) by administration of a unit dose of the antibody.

[0138] The combined administration may be co-administered using separate pharmaceutical compositions or a single pharmaceutical composition, or may be sequential in either order, optionally with a period during which both (or all) therapeutic agents simultaneously exert their biological activities. Such combined therapy may result in a synergistic therapeutic effect. In certain embodiments, it may be desirable to combine the administration of an antibody of the invention with another antibody directed against another Plasmodium falciparum antigen or against a different CSP target epitope.

[0139] In certain embodiments, the antibody can be administered by gene therapy via nucleic acid comprising one or more polynucleotides encoding the antibody. In certain embodiments, the polynucleotide encodes an scFv. In certain embodiments, the polynucleotide comprises DNA, cDNA, or RNA. In certain embodiments, the polynucleotide is present in a vector, for example, a viral vector.

[0140] Method for selecting anti-CSP antibodies as antimalarial therapeutic antibodies Based on the analysis of anti-CSP antibodies described herein, a) in vivo antibody protective activity does not correlate with binding kinetics to long NANP6 peptides (Table 9), but k off The present invention demonstrates that a) the binding activity of CSP significantly correlates with the binding rate to both a short NANP-containing peptide (NPNA3) and the minor repeat and junction region (JR) tetrapeptide (Table 9), and b) the binding activity resulting from promiscuous binding can promote a more protective response in vivo.

[0014] Provided herein is a method for selecting anti-CSP antibodies (therapeutic anti-malaria antibodies) for the prevention or treatment of malaria. In certain embodiments, the method includes analyzing antibodies for binding to a first epitope in the central repeat region of CSP and for binding to a second epitope in CSP that is heterologous to the epitope present in the RTS,S vaccine, wherein the antibody is selected if it binds to both the first epitope in the central repeat region of CSP and the second epitope that is heterologous to the epitope present in the RTS,S vaccine (heterologous epitope). In certain embodiments, the method involves selecting an antibody as an anti-malarial therapeutic antibody if the antibody binds to a first epitope in the central repeat region of CSP and to a second epitope that is heterologous to the epitope present in the RTS,S vaccine (a heterologous epitope).

[0141] In certain embodiments, the central repeat region of a CSP epitope comprises the amino acid sequence NPNA. Epitopes comprising NPNA include, for example, NPNANP, NANPNA, ANPNAN, NANPNANP, ANPNANPN, NPNANPNA, PNANPNAN, (NPNA)3, or (NPNA)4. In certain embodiments, heterologous epitopes include epitopes of the minor repeat region of CSP, including epitopes comprising DPNA / NPNV, and epitopes of the junction region of CSP, including epitopes comprising DPNA.

[0142] The CSP antibodies and variants thereof disclosed herein can be selected as antimalarial therapeutic antibodies based on their binding specificity. For example, but not limited to, the CSP antibodies and variants thereof disclosed herein can specifically bind to a first epitope (e.g., those disclosed in Table 4) and a second epitope (e.g., those disclosed in Table 5).

[0143] In certain embodiments, the method includes analyzing the antibody for binding to a first epitope of CSP. In certain embodiments, the first epitope is included in the central repeat region of CSP. In certain embodiments, the first epitope comprises the amino acid sequence set forth in SEQ ID NOs: 923-974. In certain embodiments, the first epitope consists of the amino acid sequence set forth in SEQ ID NOs: 923-974. In certain embodiments, the method also includes analyzing the antibody for binding to a second epitope of CSP. In certain embodiments, the second epitope is heterologous to an epitope present in the RTS,S vaccine. In certain embodiments, the second epitope comprises the amino acid sequence set forth in SEQ ID NOs: 975-1195. In certain embodiments, the second epitope consists of the amino acid sequence set forth in SEQ ID NOs: 975-1195. In certain embodiments, the antibody is selected as an anti-malarial therapeutic antibody if it binds to both the first epitope and the second epitope.

[0144] In certain embodiments, the method further comprises analyzing the antibody for binding to at least one additional epitope of CSP. In certain embodiments, the at least one additional epitope is heterologous to an epitope present in the RTS,S vaccine. In certain embodiments, the at least one additional epitope comprises an amino acid sequence set forth in SEQ ID NOs: 975-1195. In certain embodiments, the at least one additional epitope consists of an amino acid sequence set forth in SEQ ID NOs: 975-1195. In certain embodiments, an antibody is selected as an anti-malarial therapeutic antibody if it binds to the first epitope, the second epitope, and at least one additional epitope.

[0145] In certain embodiments, the antibody has a binding affinity to the first epitope of about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 A binding affinity (K D In certain embodiments, the antibody binds to the second epitope at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D In certain embodiments, the antibody binds to the first epitope at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D and binds to a second epitope at about 10-6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D Combine with.

[0146] In certain embodiments, the antibody has about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is less than M D In certain embodiments, the antibody further binds to the first epitope at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D and binds to the second epitope at approximately 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K is M D and binds to at least one additional epitope at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10-13 K is M D Combine with.

[0147] In certain non-limiting embodiments, K D can be measured using a surface plasmon resonance assay using a BIACORE® at 25°C with an immobilized antigen CM5 chip of approximately 10 response units (RU). Briefly, after activation of a carboxymethylated dextran biosensor chip, each epitope is diluted and injected at a consistent flow rate (e.g., 5 μl / min). After injection of the epitope, unreacted groups are blocked. The association rate (k on or k a ) and dissociation rate (k off or k d ) is calculated using a binding model that simultaneously fits the association and dissociation sensorgrams. D is the ratio k d / k a (k off / k on ) is calculated as K D Additional information regarding the calculation of can be found in Chen et al., J. Mol. Biol. 293 (1999) 865-881. * * * * *

[0148] From the above description, it will be apparent that variations and modifications may be made to the subject matter disclosed herein to adapt it to various applications and conditions. Such embodiments also fall within the scope of the following claims.

[0149] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0150] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

[0151] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0152] The foregoing written description is deemed sufficient to enable one of ordinary skill in the art to practice the methods and / or obtain the compositions described herein. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0153] The disclosures of all references herein are expressly incorporated herein by reference. [Example]

[0154] These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and are encompassed within the scope of the appended claims.

[0155] Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0156] Example 1. Identification of functionally active anti-CSP antibodies PB was biased toward the dominant mutant immunoglobulin G lineage after RTS,S vaccination Anti-CSP antibodies were discovered in antibody repertoires generated by Immune Repertoire Capture® (IRC®) technology from plasmablast B cells isolated from two donors enrolled in a Phase 2a study evaluating the efficacy of the RTS,S vaccine in preventing malaria infection. IRC® technology and its use in antibody discovery are well known and are disclosed, for example, in WO2012148497A2, the entire contents of which are incorporated herein by reference. The RTS,S vaccine is a pseudovirion vaccine that combines the central repeat and C-terminal regions of the hepatitis B surface antigen and CSP protein. RTS,S consists of two polypeptides: RTS, a single polypeptide chain corresponding to amino acids 207–395 of P. falciparum (3D7) fused to HBsAg, and S, a 226-amino acid polypeptide corresponding to HBsAg. Stoute, et al., N Engl J Med;336:86-91 (1997); RTS,S Clinical Trials Partnership, PLoS Med.11(7):e1001685, (2014), WO1993 / 10152. RTS,S vaccines were administered with the adjuvant AS01B to increase efficacy. AS01B is a liposome-based formulation containing the immunostimulants monophosphoryl lipid A (MPL) and QS21, and has been shown to be more immunogenic than another adjuvant used in earlier studies, AS02A. Kester, et al., J Infect Dis 200:337-346 (2009). All study participants were vaccinated with one of two vaccine schedules (standard full dose: 0, 1, 2M or fractional third dose: 0, 1, 7M) or placebo and then challenged with a controlled human malaria parasite infection (CHMI).

[0157] Plasmablasts (PB) were isolated from PBMCs collected prior to CHMI, 7 days after the third dose (P3D, n = 22,319 PB) and 7 days after the fourth dose (P4D, n = 10,429 PB; Table 9), and used to generate naturally paired heavy and light chain IgG sequences. Nearly all (99.2%) of the antibody sequences diverged from the predicted germline precursor sequences. Consistent with previous malaria studies, specific germline heavy and light chain genes and pairings, including IGHV3-30 / 33, KV1-5, KV3-20, and LV1-40, were frequently observed in the dataset. No significant associations were observed between protection status and multiple IgG sequence and repertoire characteristics examined.

[0158] In a Phase 2a clinical trial, immunoglobulin (Ig)G-expressing PB messenger RNA isolated from peripheral blood mononuclear cells (PBMCs) of individuals (n=45) vaccinated with RTS,S was sequenced using the Immune Repertoire Capture® sequencing platform. In this study, participants received either three full doses of RTS,S / AS01E (012M, n=15) spaced one month apart or two full doses spaced one month apart, followed by a smaller (fifth, "fractionated") dose (Fx017M, n=30) six months later. Vaccine recipients were challenged with malaria in a controlled human malaria infection (CHMI) model after the third dose. A subset received a fourth dose and a second challenge with malaria. PBs were isolated from PBMCs collected prior to CHMI, 7 days after the third dose (P3D, n = 22,319 PB) and 7 days after the fourth dose (P4D, n = 10,429 PB; Table 6), and used to generate naturally paired heavy and light chain IgG sequences. Nearly all (99.2%) of the antibody sequences diverged from the predicted germline precursor sequence (Figure 4). Consistent with previous malaria studies, specific germline heavy and light chain genes and pairings, including IGHV3-30 / 33, KV1-5, KV3-20, and LV1-40, were frequently observed in the dataset (Figures 5A-5C). No significant associations were observed between protection status and multiple IgG sequence and repertoire characteristics examined (Figures 5D-5I). [Table 6]

[0159] P3D and P4D PBs were grouped into Ig lineages (n = 18,980), defined here as PB sequences likely derived from a common precursor B cell clone (see Example 2). Lineage sizes ranged from 1 to 84 (P3D) or 1 to 93 (P4D) PBs. Because PBs have a short half-life in blood and were isolated from small volumes of blood (approximately 10 ml), the detection of lineages with more than two PBs indicates recent expansion in lymphoid organs. One-fifth of the lineages were propagated and contained at least two PBs with either the same or divergent B cell nucleotide sequences (19.4%, n = 3,684 lineages, Figure 3A). Consistent with antigen-driven selection pressure after vaccination, most of the propagated lineages also showed evidence of clonal expansion, a hallmark of affinity maturation (Figure 3B). Furthermore, several lineages had clonal representatives observed after both the third and fourth immunizations and are referred to herein as "recall" lineages (4.1% to 26.6% of vaccinee P3D-expanded lineages). Additionally, when sequences were compared between vaccinees, many of these expanded lineages also showed evidence of sequence convergence between two or more vaccinees (7.3% to 46.7% of vaccinee P3D-expanded lineages). Not surprisingly, lineages (n = 10,841) with only a single observed PB in their P3D repertoires had significantly lower convergence rates (2.0% to 13.8%) and recall rates (1.2% to 18.6%), as well as higher levels of somatic hypermutation (SHM), than expanded lineages (P < 0.0001 and P < 0.001, respectively, Wilcoxon matched-pair, two-tailed test). Therefore, to increase the likelihood of identifying antibodies originating against the RTS,S antigen, subsequent analytical efforts focused on the expanded lineages (Fig. 3B).

[0160] The lineage with the greatest number of PBs per vaccinee, herein referred to as the "dominant lineage," was hypothesized to be more likely to be targeted by the vaccine because it outperformed other PB lineages for antigen binding and / or T cell help in lymphoid organs. Therefore, for each vaccinee, the expanded P3D lineages (9–99 expanded lineages were observed per vaccinee) were ranked by size ("rank size"). The sum of the PBs in each vaccinee's top four ranked-size lineages constituted 17%–100% of the total number of PBs in each vaccinee's expanded lineage P3D repertoire and 33% of the PBs in the P3D expanded lineages from all vaccinees (Figure 3C). Because this pattern of PB distribution was consistent across protection status and dose regimens, we generated antibody screening libraries for in vitro and in vivo characterization biased toward the dominant P3D lineages in both protected and unprotected vaccinees.

[0161] CSP responsiveness of expanded P3D PBs is associated with low SHM and defective defense Clones from each of the 369 unique P3D lineages were selected, gene synthesized, and recombinantly expressed for testing (Figure 6A). This library included nearly all (96%) of the largest lineages (rank size 1) across all vaccinees, approximately half (56%) of the second-, third-, and fourth-largest lineages across all vaccinees, a small subset (6.9%) of expanded subdominant lineages (rank size 5 or greater), and several single PB lineages (0.18% of 10,841 single-cell lineages). All antibodies were screened by CSP enzyme-linked immunosorbent assay (ELISA) (Figures 3D and 6B), and approximately one-third were screened against another RTS,S component, hepatitis B surface antigen (HBsAg, Figure 6C). Of the antibodies screened by both assays (n = 130), 52% were reactive to CSP or HBsAg (67 / 130). In total, 38% (139 / 369) bound to CSP, and the binding of an additional 29 antibodies was uncertain. Of the CSP-reactive antibodies, 73% (102 / 139) bound to peptides derived from the NANP CR region, and 20 bound to peptides derived from the C-terminal region (data not shown).

[0162] Given that expanded lineages were more likely to show evidence of convergence and recall compared with single PBs, we tested whether these same characteristics were associated with CSP reactivity. Indeed, antibodies from lineages showing sequence convergence across two or more vaccinees were more likely to bind CSP (54%, 55 / 102) than clones from lineages lacking evidence of convergence (31%, 84 / 267, P = 0.0001, Fisher's exact, two-tailed). Recalled lineages were also more likely to be CSP reactive (49%, 43 / 87) compared with lineages in which only P3D was observed (19%, 16 / 83, P < 0.0001, Fisher's exact).

[0163] As previously described for immunization with whole sporozoites, SHM levels of CSP-reactive antibodies were significantly lower than those of CSP-negative antibodies (P < 0.0001, Figure 3D), and SHM levels of NANP-specific antibodies were lower than those of C-terminal binding antibodies (P < 0.006, Figure 3D). Consistent with these sequence repertoire observations, SHM levels of NANP-binding antibodies did not correlate with vaccinee protection status P3D (P > 0.6, Figure 7B). Furthermore, the percentages of antibodies that were CSP-specific and NANP-specific were surprisingly lower among vaccinees protected at P3D than among vaccinees not protected at P3D (P < 0.0007 for CSP and P < 0.006 for NANP, Fisher's exact, Figures 3E and 3F). This inverse correlation between antibody binding and P3D protection status is also observed when the analysis is restricted to only antibodies from the most dominant lineages (rank size 1-4, P<0.0004, Figure 3G) and when all antibodies, including the 20 from lineages with only 1 PB, are combined in the analysis (P<0.0005 by Fisher's exact and bootstrap analysis, P<0.00105, Figure 3H). These data suggest that the quality of the CSP-specific antibody repertoire may be more important in promoting protection than the overall amount of circulating CSP-specific or repeat-specific PB.

[0164] Sporozoite-inhibitory antibodies in P3D PB are not sufficient for P3D protection. Given this surprising inverse correlation and the well-documented protective activity of CSP-binding antibodies in both humans and mice, antibodies were selected for advancement as potential antimalarial prophylactics, without assuming any correlation of protection. Seventy-seven antibodies (77 unique lineages) were selected, including NANP-reactive and C-terminal-reactive antibodies from protected (n = 26) and unprotected (n = 8) vaccinees, as well as dominant and subdominant lineages with either high or low SHM levels (≥ 20 or < 20 nucleotide mutations from germline per antibody, respectively). Because in vitro functional assays demonstrated limited predictive power for in vivo antimalarial activity, antibodies were screened for activity using a mouse sporozoite infection model. The majority of these antibodies (44 / 77) provided greater than 95% inhibition of sporozoite liver burden, and several provided near-complete protection (≥ 99.9% inhibition). All 44 antibodies bound to the NANP repeat region of CSP, most of which were derived from the IGHV3-33 germline, several from other IGHV3 genes, and one from IGHV1. Thirteen additional NANP-binding, IGHV3-30 / 33 antibodies showed limited inhibition of parasite liver burden (80%–95%), and 12 antibodies, including three C-terminal peptide binders, showed minimal but detectable inhibition (20%–80%, Figure 1A ).

[0165] Approximately one-third (30%, 23 / 77) of the antibodies tested were from unprotected vaccinees, including half (7 / 14) that demonstrated near-complete protection in mice (>99.9% inhibition, Figure 1A). These data suggest that the expression of these inhibitory antibodies by circulating, expanded P3D PB lines is insufficient to confer protection. For example, the highly potent antibody AB-000317 is observed in both protected and unprotected vaccinees (Figure 3H, red circle). However, the PB expansion levels of this antibody lineage differ between the two vaccinees. In protected vaccinees, the antibody was expressed in members of the largest PB lineage, whereas in unprotected vaccinees, the antibody was expressed in the seventh-ranked lineage (15.8% vs. 1.7% of PB in expanded P3D lines, and 10% vs. 0.9% of all circulating P3D PB, respectively). These data are consistent with the hypothesis that, in addition to the functional activity of antibodies, the number of PBs expressing an antibody may influence the protective status by ultimately affecting its titer in the blood and / or its representation in immune memory.

[0166] Inhibitory antibodies from vaccinees bind to CSP peptides that are not present in RTS,S. To investigate the potential development of these inhibitory antibodies as potential drugs, we selected 35 NANP repeat-binding strains for further pharmacological testing from the 52 that demonstrated 90% or greater inhibition in sporozoite challenge screening. To avoid sequence features that could potentially act as a disadvantage during antibody drug development and to investigate clones from strains with extensive clonal diversity, we selected two or more unique antibody clones from several (n = 23) strains. Overall, up to 141 antibodies from 21 protected vaccinees of both RTS,S dose regimens, representing a range of high and low SHM levels, were tested in the binding assay.

[0167] The antibody exhibited a wide range of affinity for CSP (K by surface plasmon resonance [SPR]: 11 pM to 9.8 nM). D, Figure 1B, Tables 7 and 8). Despite the observed inverse correlation between the protection status of vaccinees and the percentage of CSP-reactive antibodies observed in the original screening library (Figures 3E-3H), these downselected inhibitory antibodies exhibited a significant decrease in CSP-binding affinity (K D There was a significant association between the binding association rate (k) and SHM levels (P<0.005, r=-0.26 and P<0.0001, r=-0.39 for heavy and light chains, respectively, Spearman test), indicating that affinity maturation to CSP occurred after vaccination. These correlations were due to the relationship between two factors: the binding association rate (k on ) and heavy and light chain SHM levels (Figures 10A and 10B, Table 9), as well as binding dissociation rates from CSP (k off ) and likely driven by SHM levels in the light chain (P<0.0005, r=-0.29, Spearman, Table 9). [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 8-1] [Table 8-2] [Table 9]

[0168] The antibodies were also evaluated for binding to short (12-15 residues) and long (20-24 residues) peptides derived from various tetrapeptide-based homologous (NPNA3 and NANP6 peptides) and heterologous epitopes of CSP (NPDPNANPNVDPNANP, NVDP3NANP2, and the junction [KQPADJNPDPNANPN] peptide) (Figure 1B). Among the strongest correlations observed were the SHM and k for the short major repeat peptide, short minor repeat peptide, and JR peptide. off There was an inverse correlation between K and K (Figures 1C-1E). K calculated for long homologous and heterologous peptides, as well as for CSP off The rates show either a weak but still statistically significant correlation with SHM levels, or no significant correlation at all (Figures 1F-1G, Table 9). Indeed, the strongest correlation was observed between SHM and binding rates for short homologous peptides, even though the longer versions of the homologous peptides contain more repeats of the same epitope (Table 9).

[0169] Furthermore, the correlation between SHM levels and binding rates to JR peptides, which are heterologous to the RTS,S epitope, was stronger than that for longer homologous peptides (Table 9). These data indicate that B cell receptor maturation of these highly functional antibodies may have been preferentially driven by interactions with short versus long NANP epitopes, which benefited maturation to heterologous peptide sequences. These observations are consistent with reports that protective antibodies from anti-CSP immune responses can exhibit promiscuous binding across different CSP epitopes, although other reports indicate that such promiscuity is not necessarily required for protection.

[0170] Anti-sporozoite activity correlates with CSP-peptide binding and SHM levels. Seventy antibodies, representing 33 of the 35 protective strains evaluated in the binding studies, were directly compared to the highly efficacious antibody AB-000317 in an intravenous sporozoite challenge mouse model. The antibodies inhibited 44.1% to 97.5% of sporozoite liver burden (47.4% to 103.8% of AB-000317 inhibition, Tables 7 and 8). Overall, approximately half of the antibodies showed comparable inhibition to AB-000317 (n = 32), while the other half showed significantly weaker inhibition (n = 36), and one, AB-000224, showed superior activity to AB-000317 (Figures 2A-2B, Tables 7 and 8). Serum concentrations of most antibodies were significantly higher than the CSP K of the respective antibody. D The activity of AB-000317 was at least 1000-fold higher than that of AB-000317 (Figure 2C, Tables 7 and 8), indicating that the weak inhibition was unlikely to be due to low levels of circulating antibodies. Lines with at least one antibody that showed activity consistent with AB-000317 were considered for further advancement.

[0171] To determine whether RTS,S-facilitated affinity maturation contributed to antibody inhibition, we assessed whether percent inhibition compared to AB-000317 correlated with peptide binding rate or SHM level. Relative activity was measured by the slow kDa binding rate from CSP. off (Fig. 2D), a short homologous peptide, slow k from NPNA3 off (Fig. 2E), as well as slow k from JR and other short heterologous peptides. off Surprisingly, even though this peptide is the most representative of both RTS,S and CSP, no significant correlation was observed between its inhibitory activity and its binding rate to the long homologous peptide, NANP6 (P > 0.3 [k off ];P>0.7[k on [Spearman and Pearson, Table 9]. Taken together, the data evaluating these inhibitory antibodies suggest that, although binding to the NPNA epitope may be required, mutations that favor binding to heterologous peptides may be preferable over mutations that simply improve binding to the long homologous NPNA epitope.

[0172] SHM-mediated affinity maturation likely underlies the correlation between in vivo function and binding kinetics, as inhibitory activity significantly correlated with germline-derived heavy and light chain nucleotide and amino acid changes (Figures 2H-2I, Table 9). Consistent with this observation, low-SHM antibodies were more likely to exhibit significantly weaker inhibition compared to AB-000317 than antibodies with a higher mutational burden (86% [12 / 14] vs. 44% [24 / 55], P = 0.0069, Fisher's exact, two-tailed). Collectively, these correlations between higher SHM levels and binding kinetics to homologous (Figures 1C and 1F) and heterologous epitopes (Figures 1D-1E, and 1G), as well as between higher SHM levels and inhibitory activity (Figures 2H-2I), suggest that affinity maturation to RTS,S epitopes involves bystander maturation to heterologous epitopes that may be functionally important.

[0173] SHM levels, inhibitory activity, and k from CSPs and short peptides off Despite the correlation between SHM and κ, some antibodies with high SHM levels are exceptions. In some cases, high SHM antibodies exhibit relatively fast k off and slow k on These antibodies have relatively fast kappa activity, making them relatively poor inhibitors like many of the low-SHM antibodies (Figure 2J). These antibodies may be due to inefficient affinity maturation, resulting in fewer inhibitory paratopes, and / or aberrant selection mechanisms that limit survival in and recall from memory (Figure 3E-3H). In other cases, some high-SHM antibodies have relatively fast kappa activity against short peptides. off and slow k onAlthough they have unfavorable binding kinetics, they are still relatively good inhibitors (Figure 2J). In these latter cases, affinity maturation to antibody homotypic Fab-Fab interactions, rather than to the CSP epitope, may contribute to the relatively strong activity. Antibody-antibody binding events can contribute to anti-CSP binding capacity and increased functional activity, and have been reported for some of the antibodies described herein. Such homotypic interactions may not be reflected in binding kinetics for short NPNA3 peptides, which, due to their short length, cannot sterically accommodate multiple simultaneous binding events. Indeed, the relatively fast k for short peptides off Four antibodies with activity comparable to that of AB-000317 are derived from a lineage containing antibodies that bind via Fab-Fab homotypic interactions (AB-000399, Figure 2J, red circle). Overall, the data are consistent with antibody affinity maturation through multiple distinct binding modes, revealing several antibodies (>30) with activity comparable to that of AB-000317 that could potentially be developed into clinical leads.

[0174] Using the sporozoite liver burden data, this example further selected 26 mAbs representing 15 strains for evaluation in the parasitemia burden model as a surrogate endpoint for assessing in vivo function. This set included AB-000317, AB-000224, 23 other mAbs with liver burden inhibitory activity similar to AB-000317, and one mAb with weaker activity than AB-000317. All but two mAbs were significantly more likely to prevent parasitemia than the negative control. Seven mAbs, including AB-000224 and two other mAbs from the same strain, showed a trend toward superior protection against AB-000317 (nonparametric log-rank hazard ratio <1 vs. AB-000317, Figure 2K, Tables 10-12). Serum concentrations of nearly all mAbs (25 / 26) at the time of infection were at least 1000-fold higher than the KDCSP-SPR of the respective mAb (Tables 7 and 8 ), suggesting that mAbs more effective than AB-000317 are likely not missed due to low levels of circulating antibodies. [Table 10] [Table 11] [Table 12]

[0175] Example 2. Method This example provides details of the methods and experimental strategies employed for the results illustrated in Example 1 above.

[0176] Vaccine recipients, plasmablast isolation, IgG sequencing PB collection was part of a Phase 2a clinical trial of the RTS,S / AS01 (Mosquirix™) vaccine with a split third and fourth dose, the protocol of which was approved by the Walter Reed Army Institute of Research Institutional Review Board and the Western Institutional Review Board, and written informed consent was obtained from each subject before study procedures began (ClinicalTrials.gov identifier: NCT01857869). Unique samples from study participants obtained as PBMCs for this study were used exhaustively and are not available.

[0177] Plasmablast isolation, cloning, and sequencing were performed using publicly available protocols as follows: PBMCs were stained with the following mAbs: anti-CD3-FITC (BioLegend, Cat. No. 300406, clone UCHT1), anti-CD14-FITC (BioLegend, Cat. No. 325604, clone HCD14), anti-CD19-BV421 (BioLegend, Cat. No. 302234, clone HIB19), and anti-CD20-PerCP / cy5.5 (BD, Cat. No. 340955, clone L2 7), anti-CD27-BV510 (BioLegend, catalog number 302836, clone O323), anti-CD38-PE / cy7 (BioLegend, catalog number 356607, clone HB-7), anti-IgA-FITC (Miltenyi, catalog number 130-113-175, clone IS11-8E10), anti-IgM-APC / cy7 (BioLegend, catalog number 314520, clone MHM-88). IgG+ PBs were single-cell sorted into 96-well PCR plates containing hypotonic buffer (330 nM dNTPs (NEB, catalog no. N0447L), 1 μg / ml BSA (NEB, catalog no. B9000S), 2 mM DTT (Sigma-Aldrich, catalog no. 43816), 0.5% IGEPAL-430 (Sigma-Aldrich, catalog no. I8896), and 200 units / ml Ribolock (Thermo Fisher Scientific, catalog no. EO0384)) based on gating for CD3-CD14-CD19+CD20-CD27+CD38++IgA-IgM- cells.Sequencing of IgG mRNA isolated from single-cell sorted PBs was performed with the following modifications: desthiobiotinylated oligo(dT) and Maxima H-reverse transcriptase (Thermo Fisher Scientific, catalog number EP0753) were used for reverse transcription, cDNA was extracted using Dynabeads™ MyOne™ C1 streptavidin beads (Thermo Fisher Scientific, catalog number 65001), the concentration of the final NGS library preparation was determined using qPCR (KAPA SYBR® FAST qPCR Kit for Titanium, Kapa Biosystems), and natively paired IgG heavy and light chain amplicons were sequenced using a Roche FLX+154 Titanium sequencing kit.

[0178] DNA barcode assignment and sequence assembly were performed as described: a minimum coverage of 10 reads was required for each heavy and light chain assembly to be acceptable. Both heavy and light chain reads were required to assemble a unique contig within a well. If there were more than two contigs, the well was rejected from consideration unless one of the contigs contained at least 90% of the reads.

[0179] Analysis of sequence, phylogenetic, and repertoire characteristics Determination of germline assignment and SHM levels Assignment of variable (V), diversity (D), and joining (J) gene segments and identification of mutations were performed using the Somatic Diversity Analysis (SoDA)65 implementation and the IMGT Human Immunoglobulin Germline Database release, IMGT_20203166. SHM substitutions were counted for each antibody by aligning the heavy and light variable domains (start of framework 1 to end of framework 4) with a hidden Markov model that includes the germline alignment region (VDJ for heavy, VJ for light) and the state of the N-nucleotide region, counting substitutions relative to the germline sequence only in the aligned portion (not including rare observed indels). IgG isotype assignment (IgG1-4) was performed by aligning the sequence 3' of framework 4 to the IMGT human Ig constant region sequence from IMGT_20203166.

[0180] CDR3 and lineage assignment The complementarity-determining region 3 (CDR3) sequence was defined by the first amino acid residue of the Kabat annotation plus framework 4, from which the CDR3 length was calculated. Naturally paired IgG sequence clones were assigned to the same lineage if they were derived from the same vaccinee, had the same IGHV and IGK / LV germline gene assignment, the same heavy chain CDR3 (H3) length, the same light chain CDR3 (L3) length, and at least 75% nucleic acid sequence identity across the combined H3 and L3. In some cases, clones with IGHV3-33 and IGHV3-30 (germline genes with high sequence identity) met all criteria for being assigned to the same lineage, except for the IGHV. In these cases, clones were assigned to the same lineage. Lineages were assigned rank sizes based on the lineage frequency (the number of PBs expressing a clone in a lineage divided by the total number of PBs in the repertoire). In some cases, two or more lineages in a repertoire have the same rank size because they have the same number of PBs.

[0181] Convergence, clonality, and recall Two IgG clones were defined as convergent if they originated from different vaccinees, had the same IGHV and IGK / LV germline gene assignments, the same H3 length, the same light chain CDR3 (L3) length, and at least 85% BLOSUM62-weighted amino acid sequence identity between the concatenated H3 and L3. A lineage was defined as convergent with another lineage if it originated from different vaccinees and at least one IgG clone was present in the first lineage that converged with at least one IgG clone in the second lineage. Clonality was summarized as the normalized entropy across all lineages in each P3D vaccinee repertoire. Specifically, it is the sum of -(Ki / N*log(Ki / N))) / log(N), where i spans 1...N, where N is equal to the number of lineages in the repertoire, Ki is the size of each lineage as the number of PBs, and i ranges from 1 to N. Normalized entropy takes values ​​between 0 and 1 inclusive, where 0 means a single lineage is completely dominant and 1 means a set of N>1 several lineages are all equally abundant. Recalled lineages were defined as lineages with at least one PB antibody clone observed in both the P3D and P4D repertoires among vaccinees (n=17) in which at least 100 PBs were sequenced from P4D PBMC samples.

[0182] Lineage and clone selection for CSP ELISA screening library PB lineages (n=369) for initial screening of CSP reactivity were selected as described in Example 2, and specific clones from each selected lineage were selected for recombinant expression and screening based on one or more of the following characteristics: i) the clone has paired heavy and light chain amino acid sequences expressed by more PBs in the lineage than any other clones ("dominant clone"), and / or ii) the selected specific antibody sequence converges with at least one other specific antibody sequence in another vaccinee's lineage, with convergence defined by the method described in "Convergence, Clonality, and Recall" ("convergent clone"), and / or iii) the clone is identified by the "leafiest lineage" in a phylogenetic tree of lineages ranked according to the number of leaves with the largest terminal clade, where the terminal clade of the leaf is defined as the most leafy ("leafiest lineage clone"), and / or iv) the clone has the highest number of germline-derived nucleic acid mutations among all clones in the lineage ("most mutated clone"). Three clones (0.8% of the screening library) did not meet any of these criteria due to errors that were not detected until after the screening was performed. The proportions of antibodies meeting each criterion from protected and unprotected vaccinees were not statistically different (Fisher's exact) from the proportions of all antibodies in the screening library.

[0183] Recombinant antibody production Each antibody gene sequence was cloned into a high-expression mammalian vector. Briefly, variable region sequences were synthesized and subcloned into expression vectors containing human IgG1 heavy chain and the appropriate human kappa or lambda light chain constant region coding domain sequences. Each completed construct was sequence verified before proceeding to DNA plasmid production scale-up. Suspension HEK293 cells were seeded in shake flasks and grown using serum-free, chemically defined medium. On the day of transfection, the grown cells were seeded into new flasks with fresh medium. Each DNA construct was transiently transfected into HEK293 cells using the cationic lipid transfection method. Cells were maintained as batch-fed cultures until the end of the production run. Conditioned medium from the transient production run was collected and clarified by centrifugation and filtration. The supernatant was loaded onto a Protein A column pre-equilibrated with binding buffer. Wash buffer was passed through the column until the OD280 value (NanoDrop, Thermo Scientific) measured zero. The target protein was eluted with a low pH buffer, fractions were collected, and the OD280 value of each fraction was recorded. Fractions containing the target protein were pooled and filtered through a 0.2 μm membrane filter. The purified antibody was dialyzed against PBS and analyzed using a LabChip GXII. Endotoxin measurements were performed using a chromogenic Limulus Amebocyte Lysate method with Pyrochrome (Associates of Cape Cod).

[0184] CSP, NANP peptide, and C-terminal peptide ELISA Antibodies were mapped to CSP using the nearly full-length CSP, (NANPx6) peptide, and the CSP C-terminal peptide (Pf16). For the purposes of this study, antibodies were classified as "positive," "negative," or "indeterminate." All antibodies were evaluated at concentrations of either 0.15 or 0.04 μg / ml. ELISA optical density (OD) values ​​were converted to fold induction relative to the average of four negative control antibodies run in each experiment. The range of OD responses observed in each experiment was then used to determine the boundary "indeterminate" range for that experiment. Antibody ODs that did not exceed the mean negative control antibody OD + 3-fold standard deviation were classified as "negative." Antibodies with ODs above the mean negative control antibody OD + 3-fold standard deviation but not yet exceeding 20% ​​of the negative threshold + OD range for the experiment were classified as "indeterminate." Any antibody ODs above the experimental "indeterminate" threshold were classified as "positive."

[0185] HBsAg ELISA The MONOLISA Anti-HBs EIA kit [Bio-Rad catalog number 25220] was used to determine the HBsAg reactivity of antibodies. A four-point 1:3 dilution series was prepared in duplicate for each tested object. The maximum stock input was 10% of the total purified volume for each of the 139 tested antibodies. If less than 10% of the total purified volume was required, the starting concentration of each tested antibody was individually adjusted to 300 nM. Otherwise, the starting concentration was based on the amount of protein contained in 10% of the total purified volume. The cutoff calibrator from the kit was run in quadruplicate, and both negative controls were run in duplicate. A four-point, 1:3 dilution dose-response curve of PC3, starting at 150 pM, was also run in duplicate. An antibody was considered HBsAg positive if the signal met the cutoff calibrator criterion for at least one concentration below 30 nM. An antibody was considered "borderline" HBsAg reactive if the signal was negative at concentrations tested below 30 nM but met the cut-off calibrator criteria for any concentration above 30 nM. An antibody was considered negative if the signal did not meet the cut-off calibrator criteria for the concentrations tested.

[0186] Selection of mAbs for initial characterization in a mouse sporozoite challenge model Of the 102 antibodies that were reactive in the NANP6 peptide ELISA (Figure 1D), 69 were originally selected for in vivo screening based on IGHV expression, vaccine protection status, and level of SHM. Specifically, the 102 antibodies were divided into 11 groups based on the 11 different IGHVs expressed among them (IGHV1-2, 1-69, 1-8, 3-15, 3-23, 3-30, 3-33, 3-48, 3-49, 3-7, and 5-51). At least half of the mAbs in each set were selected, including mAbs from both the protected (n = 26) and unprotected (n = 8) vaccines. Only antibodies with high levels of SHM (≥ 20 nucleotide mutations from the germline per antibody) were selected, except for the set containing IGHV3-33 and 3-49, which also contained several antibodies with low levels of SHM (< 20 nucleotide mutations from the germline per antibody). Two antibodies from these selections did not express enough material to be tested in vivo (the only antibody that contained IGHV3-23, and one of the two antibodies that contained IGHV5-51).

[0187] Of the 20 antibodies reactive in the C-terminal (Pf16) peptide ELISA, 11 of the 12 derived from protective vaccines were originally selected for in vivo screening. These included all of the IGHV germline genes observed in C-terminal (Pf16) binders from protected vaccinees (IGHV3-11, IGHV3-21, IGHV3-30, IGHV3-48, and IGHV4-59). One antibody from these selections did not express enough material to be tested in vivo (the only mAb contained IGHV3-11).

[0188] Selection of antibodies for libraries used in surface plasmon resonance (SPR) binding assays Of the 52 mAbs representing 52 unique lineages that showed 90% or greater inhibition in the initial sporozoite liver challenge mouse model screen, all from protected vaccinees (n = 36) were selected for further binding analysis, except for one mAb (AB-000239) that was observed to be reactive in the HBsAg ELISA. For each of these 35 antibodies representing 35 unique lineages, the original hit antibody was selected if it did not contain high-risk disadvantages (i.e., odd numbers of cysteines in the CDRs, any canonical N-linked glycosylation sites in the CDRs, Fv net charge (pH 5.5) > 9, or hydrophobicity index > 6.5). If the original hit had one or more disadvantages, additional clones were selected from lineages derived from either the P3D or P4D PB repertoire. In addition, two or more clones were selected from lineages with extensive inter-clonal sequence diversity. This was done using the following algorithm: i) query each clone in the lineages in order of most leafy lineage, ii) skip clones with high risk of disadvantage > 0, iii) skip any clones that are too close to any already selected clones, using the distance between clones determined as the percentage of CDR amino acids that are not conserved between clones (≤ 0) using the BLOSUM62 matrix, and iv) adjust the allowable distance between clones so that a total of 141 clones were finally selected from the 35 lineages.

[0189] High-throughput SPR Binding kinetics measurements of antibody interactions with CSP antigens were performed at 25 °C using a Carterra LSA high-throughput SPR platform and a CMD200M sensor chip (Carterra). The antigen panel included recombinant CSP and synthetic peptides NPNA3 (NPNANPNANPNA), NANP6 (NANPNANPNANPNANPNANPNANP), conjugated peptides (KQPADGNPDPNANPN), NPDPNANP2NVDP (NPDPNANPNVDPNANP), and NVDP3NANP2 (NVDPNANPNVDPNANPNVDP), which were custom-made by CPC Scientific. With the exception of NANP6, all other peptides were acetylated at the N-terminus and amidated at the C-terminus. NANP6 contained an N-terminal biotin-aminohexanoic acid tag and an unmodified C-terminus. Two microfluidic modules, a 96-channel printhead (96PH) and a single flow cell (SFC), were used to deliver liquid to the sensor chip. A single analyte antigen was titrated in each assay against the immobilized antibody.

[0190] Immobilization of antibodies to the CMD200M chip depended on the type of analyte used during titration. In assays containing recombinant CSP as the analyte, goat anti-human IgG Fc antibody (Millipore) was first immobilized on the chip via amine coupling. The chip was first activated with 100 mM N-hydroxysuccinimide (NHS) and 100 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (GE Healthcare, 1:1:1 mixed with 0.1 M MES buffer, pH 5.5) for 400 seconds, followed by immobilization of 50 μg / ml anti-human IgG Fc (in 10 mM sodium acetate, pH 4.5) for 900 seconds. Unreacted esters were quenched with a 400-second injection of 1 M ethanolamine-HCl, pH 8.5. The chip was then exposed to a double pulse (30 seconds per pulse) of 10 mM glycine at pH 2.0. CSP-specific antibodies were then captured on the anti-Hu IgG Fc surface by injecting antibodies at concentrations of 10 μg / ml or 5 μg / ml for 400 seconds using 96PH, with 1× HBSTE buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.01% Tween-20) as the running buffer and antibody diluent. When CSP peptide antigens were used as analytes, the chip was activated with NHS / EDC for 400 seconds, followed by direct immobilization of CSP-specific antibodies (in 10 mM sodium acetate, pH 4.5) injected for 400 seconds using 96PH. Unreacted esters were then quenched with a 400-second injection of 1 M ethanolamine-HCl, pH 8.5. Subsequently, nonspecifically bound IgG was washed off the sensor chip surface overnight using 45 cycles of 1x HBSTE buffer injections, also using 1x HBSTE as the running buffer, without using a regeneration buffer. Except for antibody capture with anti-human IgG Fc and washing of nonspecifically bound IgG, the running buffer was 10 mM MES buffer, pH 5.5, containing 0.01% Tween-20. Unless specified above, procedures were performed using SFC.

[0191] During the initial screening, each antibody at a given dilution was immobilized on two separate spots on the same chip, allowing duplicate measurements of binding kinetics. For binding measurements of engineered variants, each antibody was immobilized on three different spots, allowing triplicate measurements.

[0192] Two-fold dilution series of antigens were prepared in 1x HBSTE buffer. The highest concentration for full-length CSP and all CSP-peptide antigens was 8 μg / ml (0.25 μM for CSP, 2.92 μM for NANP6, 6.41 μM for NPNA3, 3.76 μM for NVDP3NANP2, 4.70 μM for NPDPNANPNVDPNANP, and 5.03 μM for N-interface). Different concentrations of antigen were then injected over the chip surface using SFC, from lowest to highest concentration, without regeneration, including eight buffer injections before the lowest non-zero concentration for signal stabilization. For each concentration, data collection included a 120-second baseline step and a 900-second dissociation step. The association step duration was 240 seconds for full-length CSP and NANP6 antigens and 300 seconds for all other CSP peptide antigens. For all assays, the running buffer for the titration was 1x HBSTE.

[0193] The collected kinetic titration data were first preprocessed with NextGenKIT (Carterra) software, including reference subtraction, buffer subtraction, and data smoothing. The data were then exported and analyzed using the in-house developed TitrationAnalysis tool. The specific binding time course of each antibody construct immobilized on a different spot was fitted to a 1:1 Langmuir model to derive ka ("k"), kd ("koff"), and KD values. KD values ​​determined for antigens with multiple repeats of the epitope include avidity effects. The average value of duplicate measurements was reported for each antibody-antigen pair from the initial screening panel. For engineered variants, the average value of triplicate measurements was reported with the following data acceptance criteria: i) the standard error of the estimated k, koff, and KD in each replicate was 20% or less, and ii) the fold change of all three parameters within the triplicate was 3 or less.

[0194] In vivo functional assessment Mouse assays were performed in strict accordance with the recommendations in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Protocols were approved by the Johns Hopkins University Animal Care and Use Committee. Protocol numbers MO18H419 and MO21H417.

[0195] Sporozoite-challenged liver burden mouse model An initial functional screen was performed in mice immunized with anti-CSP antibodies and challenged intravenously 16 hours later with 2,000 P. berghei transgenic sporozoites expressing the intact P. falciparum CSP. After 42 hours, mice were euthanized, their livers excised, RNA extracted, and transgenic 18S rRNA was measured by RTqPCR using the forward primer 5'-TGGGAGATTGGTTTTGACGTTTATGT-3' and the reverse primer 5'-AAGCATTAAATAAAGCGAATACATCCTTAC-3'. Parasite burden was expressed as P. berghei 18S rRNA copy number, and percent inhibition of burden was calculated compared to the negative control.

[0196] All other liver burden assays were performed as described by Flores-Garcia et al. 20. Briefly, Anopheles stephensi mosquitoes infected with transgenic P. berghei sporozoites expressing P. falciparum CSP and luciferase were maintained in an incubator at 19°C. Sporozoites from mosquitoes were harvested in HBSS-FBS 2% on days 20–23 postinfection. Mice were administered 100 μg of antibody per mouse (passively immunized) and challenged 16 hours later with 2,000 sporozoites injected intravenously. Control mice received either an irrelevant antibody or no antibody. 42 hours after challenge, mice were injected with 100 μl of D-luciferin (30 mg / ml), anesthetized with isoflurane, and bioluminescence expressed by the parasite in the liver was measured using an IVIS Spectrum imaging system (Perkin Elmer). The results are expressed as photons per second.

[0197] Mosquito bite-challenged parasitemia mouse model Mosquito bite challenges to assess sterilization protection were performed as described by Flores-Garcia et al. Briefly, 7- to 8-week-old mice were passively immunized with 150 μg / mouse of the indicated antibodies. 16 hours later, the mice were anesthetized and placed for 10 minutes on a cage containing five mosquitoes infected with P. falciparum CSP and luciferase-expressing P. berghei sporozoites. Four to 10 days after challenge, Giemsa-stained blood smears were examined under a light microscope to determine the appearance of parasitemia. Control mice receiving irrelevant or no antibodies were similarly challenged.

[0198] Assessment of mAb concentration in serum samples The capture antibody (AffiniPure Mouse Anti-Human IgG Fc Fragment Specific, Jackson ImmunoResearch #209-005-098) was adsorbed onto a 96-well polystyrene microplate (Immuno Plate Maxisorp, ThermoFisher Scientific #439454) in PBS (calcium- and magnesium-free Dulbecco's phosphate-buffered saline, sterile pH 7.4, Wisent #311-425-LL) overnight at 21°C, then washed three times with wash buffer (0.0.05% TWEEN 20 in PBS [Sigma #P2287]). The microplate was blocked with assay buffer (1% bovine serum albumin [Blocker BSA, ThermoFisher #37525] in wash buffer) for 1 hour at 21°C. After washing three times with wash buffer, serum samples and control standards from mice were added in duplicate in serial dilutions in normal mouse serum, then further diluted 100-fold in assay buffer and incubated for 1 hour at 21°C. The control standard consisted of AB-000317 serially diluted in 1.6-fold increments from 0.146 to 25.6 μg / ml. The microplate was then washed three times with wash buffer and incubated with horseradish peroxidase-conjugated mouse monoclonal anti-human IgG antibody (HRP-conjugated clone JDC-101, Southern Biotech #9040-05) in assay buffer for 1 hour at 21°C. After washing three times with wash buffer, the peroxidase substrate TMB (Bio-Rad #1721068) was added, followed by the addition of stop solution (TMB stop solution [650 nm], Southern Biotech #0413-01L). Absorbance was measured at 650 nm (Molecular Devices microplate reader using SoftMaxPro GxP version 6.5.1), and the concentration of human IgG in the test samples was calculated using a standard curve generated from the control antibody by interpolation of OD values ​​against a five-parameter logistic standard curve (derived from the mean OD of duplicate standard samples) and adjusted according to their corresponding dilution factors.The final sample concentration was then determined by calculating the mean of all the concentrations of the samples obtained within the range of the standard curve.

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Claims

1. A recombinant anti-circumsporozoite (CSP) antibody, wherein the recombinant anti-circumsporozoite (CSP) antibody binds to a first epitope present in the central repeat region of CSP and binds to a second epitope of CSP.

2. The recombinant antibody of claim 1 , wherein the first epitope comprises the amino acid sequence NPNA.

3. The recombinant antibody of claim 1 or 2, wherein the first epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 923 to 974.

4. The recombinant antibody of any one of claims 1 to 3, wherein the second epitope is heterologous to an epitope present in the RTS,S vaccine.

5. The recombinant antibody according to any one of claims 1 to 4, wherein the second epitope comprises a minor repeat region of a CSP and / or a junction region of a CSP.

6. The recombinant antibody of claim 5 , wherein the second epitope comprises a DPNA / NPNV-containing minor repeat amino acid sequence and / or a DPNA / NPNV-containing junction amino acid sequence.

7. The recombinant antibody according to any one of claims 1 to 6, wherein the second epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975 to 1195.

8. The recombinant antibody of any one of claims 1 to 7, wherein the antibody binds to at least one additional epitope of CSP.

9. The recombinant antibody of claim 8, wherein the at least one additional epitope comprises a DPNA / NPNV-containing minor repeat amino acid sequence and / or a DPNA / NPNV-containing junction amino acid sequence.

10. The recombinant antibody of claim 8 or 9, wherein the at least one additional epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975 to 1195.

11. The recombinant antibody of any one of claims 1 to 7, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 461.

12. The recombinant antibody of any one of claims 1 to 7, comprising a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 461.

13. The recombinant antibody of any one of claims 1 to 7, comprising a light chain variable region (VL) comprising an amino acid sequence that is at least about 80% identical to an amino acid sequence selected from SEQ ID NOs: 462 to 922.

14. A recombinant antibody according to any one of claims 1 to 7, comprising a VL comprising an amino acid sequence selected from SEQ ID NOs: 462 to 922.

15. VH comprising an amino acid sequence that is at least about 80% identical to an amino acid sequence selected from SEQ ID NOs: 1-461; A recombinant antibody according to any one of claims 1 to 7, comprising: a VL comprising an amino acid sequence that is at least about 80% identical to an amino acid sequence selected from SEQ ID NOs: 462 to 922.

16. VH comprising an amino acid sequence selected from SEQ ID NOs: 11 to 461; A recombinant antibody according to any one of claims 1 to 7, comprising: a VL comprising an amino acid sequence selected from SEQ ID NOs: 462 to 922.

17. comprising a heavy chain variable region (VH) and a light chain variable region (VH); the heavy chain variable region comprises CDR1, CDR2, and CDR3 of the heavy chain variable sequences set forth in SEQ ID NOs: 1-461; The recombinant antibody of any one of claims 1 to 7, wherein the light chain variable region comprises CDR1, CDR2, and CDR3 of the light chain variable sequences set forth in SEQ ID NOs: 462 to 922.

18. The recombinant antibody according to any one of claims 1 to 7, comprising a heavy chain variable region (VH) as set forth in Table 3 and a light chain variable region (VH).

19. The recombinant antibody of any one of claims 1 to 18, wherein the antibody exhibits at least a 20% reduction in parasite liver burden compared to a reference antibody.

20. The recombinant antibody of any one of claims 1 to 19, wherein the antibody exhibits at least a 20% increase in survival rate compared to a reference antibody.

21. The recombinant antibody of any one of claims 1 to 20, wherein the antibody exhibits increased conformational stability compared to a reference antibody.

22. The recombinant antibody of any one of claims 1 to 20, wherein the antibody exhibits increased colloidal stability compared to a reference antibody.

23. 23. The recombinant antibody of any one of claims 20, 21, or 22, wherein the reference antibody is AB-000317, AB-000224, or AB-007088.

24. A polynucleotide encoding the antibody of any one of claims 1 to 23.

25. An expression vector comprising the polynucleotide of claim 24.

26. 25. A host cell comprising the polynucleotide of claim 24 or the expression vector of claim 22.

27. A composition comprising an antibody according to any one of claims 1 to 23.

28. 28. The composition of claim 27, further comprising a pharmaceutically acceptable carrier.

29. 24. A method for preventing or treating malaria in a subject in need thereof, comprising administering an effective amount of an antibody according to any one of claims 1 to 23.

30. 30. A method of preventing or treating malaria in a subject in need thereof, comprising administering an effective amount of the composition of claim 27 or 28.

31. 31. The method of claim 29 or 30, wherein the patient is a pediatric patient.

32. A recombinant antibody according to any one of claims 1 to 23, or a composition according to claim 27 or 28, for use in the prevention or treatment of malaria in a subject in need thereof.

33. 33. The recombinant antibody or composition for use according to claim 32, wherein the patient is a pediatric patient.

34. 29. Use of a recombinant antibody according to any one of claims 1 to 23 or a composition according to claim 27 or 28 for the manufacture of a medicament for preventing or treating malaria in a subject in need thereof.

35. 35. The use of claim 34, wherein the patient is a pediatric patient.

36. 1. A method for selecting an antibody as an anti-malarial therapeutic antibody, said method comprising: a) analyzing the antibody for binding to a first epitope in the central repeat region of CSP; b) analyzing the antibody for binding to a second epitope of a CSP that is heterologous to the epitope present in the RTS,S vaccine; The method of claim 1, wherein the antibody is selected if it binds to both the first epitope and the second epitope.

37. c) analyzing the antibody for binding to at least one additional epitope of a CSP that is heterologous to an epitope present in the RTS,S vaccine; 37. The method of claim 36, wherein the antibody is selected if it binds to the first epitope, the second epitope, and the at least one additional epitope.

38. 1. A method for selecting an antibody as an anti-malarial therapeutic antibody, said method comprising: The antibody i) the antibody binds to a first epitope in the central repeat region of CSP; and ii) selecting if said antibody binds to a second epitope that is heterologous to the epitope present in the RTS,S vaccine.

39. 1. A method for selecting an antibody as an anti-malarial therapeutic antibody, said method comprising: The antibody i) the antibody binds to a first epitope in the central repeat region of CSP; ii) the antibody binds to a second epitope that is heterologous to the epitope present in the RTS,S vaccine; and iii) selecting if said antibody binds to at least one additional epitope that is heterologous to an epitope present in said RTS,S vaccine.

40. 40. The method of any one of claims 36 to 39, wherein the first epitope comprises the amino acid sequence NPNA.

41. 41. The method of claim 40, wherein the first epitope consists of an amino acid sequence selected from SEQ ID NOs: 923-974.

42. 42. The method of any one of claims 36 to 41, wherein the second epitope is heterologous to an epitope present in the RTS,S vaccine.

43. 43. The method of claim 42, wherein the second epitope comprises a minor repeat region of a CSP and / or a junction region of a CSP.

44. 44. The method of claim 43, wherein the second epitope comprises a DPNA / NPNV-containing minor repeat amino acid sequence and / or a DPNA / NPNV-containing junction amino acid sequence.

45. 45. The method of any one of claims 42 to 44, wherein the second epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975 to 1195.

46. The method of any one of claims 36 to 45, wherein the first epitope consists of an amino acid sequence selected from SEQ ID NOs: 923 to 974, and the second epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975 to 1195.

47. The antibody binds to the first epitope with a binding affinity of about 10 －６ Less than M, about 10 －７ Less than M, about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ Binding affinity (K Ｄ The method according to any one of claims 36 to 46, wherein the binding is performed by

48. The antibody binds to the second epitope with a binding affinity of about 10 －６ Less than M, about 10 －７ Less than M, about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ K is less than M Ｄ The method according to any one of claims 36 to 47, wherein the binding is carried out by

49. The antibody a) about 10 －６ Less than M, about 10 －７ Less than M, about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ K is less than M Ｄ Combined with b) about 10 －６ Less than M, about 10 －７ Less than M, about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ K is less than M Ｄ The method according to any one of claims 36 to 48, wherein the binding is carried out by

50. a) the first epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 923-974; b) the second epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975-1195; and c) the at least one additional epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975-1195.

51. The antibody binds to the at least one additional epitope in an amount of about 10 －６ Less than M, about 10 －７ Less than M, about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ K is less than M Ｄ The method of claim 50, wherein the binding is performed by

52. The antibody a) about 10 －６ Less than M, about 10 －７ Less than M, about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ K is less than M Ｄ and combine them with b) about 10 －６ Less than M, about 10 －７ Less than M, about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ K is less than M Ｄ Combined with c) about 10 to said at least one additional epitope －６ Less than M, about 10 －７ Less than M, about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ K is less than M Ｄ The method according to any one of claims 36 to 48, wherein the binding is carried out by

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