Anti-CSP antibody variants

Abstract

The present disclosure provides anti-cyclosporozoite (CSP) antibodies, compositions comprising such antibodies. Methods of producing the disclosed antibodies and methods of treating or preventing malaria using the antibodies are also disclosed. Methods of selecting antibodies as anti-malarial therapeutic antibodies are also disclosed.

Description

[0001] Cross Reference to Related Applications

[0002] 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 the provisional applications are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0003] The present disclosure relates to compositions for treating or preventing malaria, and antibodies that confer protection against infection by the malaria parasite, such as Plasmodium falciparum, transmitted by insect vectors. The present disclosure also relates to methods for treating, preventing, or diagnosing Plasmodium infection in a mammal. BACKGROUND

[0004] Malaria causes a significant burden of morbidity and mortality, especially in developing countries. The causative agent of malaria is a protozoan parasite that is transmitted by mosquitoes. Several species of the infectious Plasmodium genus cause malaria, with the most deadly being Plasmodium falciparum. Others include Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Over 80% of deaths from malaria are children under the age of five. The most advanced vaccine, and the only one recommended for use by the WHO, RTS,S / AS01 (Mosquirix®), targets the circumsporozoite protein (CSP) of P. falciparum, the species of malaria that is primarily responsible for mortality in Africa. Three immunizations with RTS,S / AS01 induce antibodies against P. falciparum CSP that act by binding to sporozoites, the infectious form of the malaria parasite introduced by a mosquito bite, and inhibiting their initial infection of liver cells. However, the immune response induced in children by Mosquirix® TM is only 45% against clinical malaria after the first dose, waning to 36% over a 4-year follow-up. Thus, other immunization approaches will be needed to achieve the WHO’s goal of reducing malaria case incidence and mortality by 90% by 2030. TM

[0005] ​Recent reports suggest that treatment with mAbs can completely prevent malaria after controlled infection and provide 88% efficacy in endemic areas for 6 months (prevention of infection). Thus, mAbs with a persistence of 4 to 6 months can provide an intervention with greater protective efficacy than seen with RTS, S, which significantly contributes to prevention of seasonal transmission. mAbs tested in clinical trials (L911 and CIS438,9) were isolated from B cells of vaccinees immunized with whole sporozoites and can prevent malaria infection by targeting specific epitopes on CSP. In view of published support for the use of mAbs for prevention as a strategy against malaria, and the successful advances of mAbs as treatments and prevention of infectious diseases in general, there is a need to generate additional protective anti-CSP antibodies with improved therapeutic characteristics. SUMMARY

[0006] The present disclosure provides anti-circumsporozoite (CSP) antibodies, compositions comprising such antibodies. Methods of producing the disclosed antibodies and methods of using the antibodies to treat or prevent malaria are also disclosed. Methods of selecting antibodies as anti-malaria therapeutic antibodies are also disclosed.

[0007] 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 the group consisting of SEQ ID NOs: 923 to 974.

[0008] In certain embodiments, the second epitope is heterologous to the epitope present in the RTS, S vaccine. In certain embodiments, the second epitope comprises the minor repeat region of CSP and / or the junction region of CSP. In certain embodiments, the second epitope comprises a minor repeat amino acid sequence comprising DPNA / NPNV and / or a junction amino acid sequence comprising DPNA / NPNV. In certain embodiments, the second epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975 to 1195.

[0009] 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 minor repeat amino acid sequence comprising DPNA / NPNV and / or a junction amino acid sequence comprising DPNA / NPNV. 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 to 1195.

[0010] 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 the group consisting of SEQ ID NOs: 1-461. In certain embodiments, the recombinant antibody comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-461.

[0011] In certain embodiments, the recombinant antibody comprises 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 the group consisting of SEQ ID NOs: 462-922. In certain embodiments, the recombinant antibody comprises a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 462-922.

[0012] In certain embodiments, the recombinant antibody comprises: a 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: 1-461; and a VL 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: 462-922. In certain embodiments, the recombinant antibody comprises: a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-461; and a VL comprising an amino acid sequence selected from SEQ ID NOs: 462-922.

[0013] 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 a heavy chain variable sequence listed in SEQ ID NOs: 1-461, and the light chain variable region comprises CDR1, CDR2, and CDR3 of a light chain variable sequence listed in SEQ ID NOs: 462-922. In certain embodiments, the recombinant antibody comprises a heavy chain variable region (VH) and a light chain variable region (VH) listed in Table 3.

[0014] In certain embodiments, the antibody exhibits at least 20% reduction in parasite liver burden compared to a reference antibody. In certain embodiments, the antibody exhibits at least 20% increase in survival compared to a reference antibody. In certain embodiments, the antibody exhibits increased conformational stability compared to a reference antibody. In certain embodiments, the antibody exhibits increased colloidal stability compared to a reference antibody. In certain embodiments, the reference antibody is AB-000317, AB-000224, or AB-007088.

[0015] In certain non-limiting embodiments, the present disclosure relates to polynucleotides encoding the presently disclosed antibodies. In certain non-limiting embodiments, the present disclosure relates to expression vectors and / or host cells comprising the presently disclosed polynucleotides.

[0016] In certain non-limiting embodiments, the present disclosure also relates to compositions comprising the presently disclosed antibodies. In certain embodiments, the compositions further comprise a pharmaceutically acceptable carrier.

[0017] In certain non-limiting embodiments, the present disclosure relates to methods of preventing or treating malaria in a subject in need thereof, the method comprising administering an effective amount of the presently disclosed antibodies or compositions. In certain embodiments, the patient is a pediatric patient.

[0018] In certain non-limiting embodiments, the present disclosure relates to methods of selecting an antibody as an anti-malaria therapeutic antibody. In certain embodiments, the method comprises: a) analyzing binding of the antibody to a first epitope of a central repeat region of CSP; and b) analyzing binding of the antibody to a second epitope that is heterologous to an epitope present in an RTS,S vaccine; wherein the antibody is selected if the antibody binds to both the first epitope and the second epitope. In certain embodiments, the method further comprises: c) analyzing binding of the antibody to at least one additional epitope of CSP that is heterologous to an epitope present in the RTS,S vaccine; wherein the antibody is selected if the antibody binds to the first epitope, the second epitope, and the at least one additional epitope

[0019] In certain non-limiting embodiments, the present disclosure relates to methods of selecting an antibody as an anti-malaria therapeutic antibody. In certain embodiments, the method comprises: selecting the antibody if i) the antibody binds to a first epitope of a central repeat region of CSP, and ii) the antibody binds to a second epitope that is heterologous to an epitope present in an RTS,S vaccine. In certain embodiments, the method comprises: selecting the antibody if i) the antibody binds to a first epitope of a central repeat region of CSP; ii) the antibody binds to a second epitope that is heterologous to an epitope present in an RTS,S vaccine; and iii) the antibody binds to at least one additional epitope that is heterologous to an epitope present in the RTS,S vaccine.

[0020] 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 the group consisting of 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 minor repeat region of CSP and / or a junction region of CSP. In certain embodiments, the second epitope comprises a minor repeat amino acid sequence comprising DPNA / NPNV and / or a junction amino acid sequence comprising DPNA / NPNV. In certain embodiments, the second epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975-1195. In certain embodiments, the first epitope consists of an amino acid sequence selected from the group consisting of 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.

[0021] In certain embodiments, the antibody binds to the first epitope with a binding affinity (K -6 M or less than about 10 -7 M or less than about 10 -8 M or less than about 10 -9 M or less than about 10 -10 M or less than about 10 -11 M or less than about 10 -12 M or less than about 10 -13 M or less than about 10 D ) to the first epitope. In certain embodiments, the antibody binds to the first epitope with a binding affinity (K -6 M or less than about 10 -7 M or less than about 10 -8 M or less than about 10 -9 M or less than about 10 -10 M or less than about 10 -11 M or less than about 10 -12 M or less than about 10 -13 M or less than about 10 D ) to the second epitope. In certain embodiments, the antibody binds to the second epitope with a binding affinity (K -6 M or less than about 10 -7 M or less than about 10 -8 M or less than about 10 -9 M or less than about 10 -10 M or less than about 10 -11 M or less than about 10 -12 M or less than about 10 -13 M or less than about 10 D ) to the first epitope; and to the second epitope with a binding affinity (K -6 M or less than about 10 -7 M or less than about 10 -8 M or less than about 10 -9M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 K of M D binds to the second epitope.

[0022] In certain embodiments, 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.

[0023] In certain embodiments, the antibody binds to the first epitope with a Kd of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 K of M D binds to the at least one additional epitope. In certain embodiments, the antibody: a) binds to the first epitope with a Kd of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 K of M D binds to the second epitope; and c) binds to the at least one additional epitope with a Kd of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 K of M D binds to the second epitope; and c) binds to the at least one additional epitope with a Kd of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12M or less than about 10 -13 K of M D binds to at least one additional epitope. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figures 1A to 1G Functional antibodies are shown to bind to CSP-derived peptides not present in the RTS, S vaccine. Figure 1A Percent inhibition in a liver stage mouse model of sporozoites is shown, and the number of nucleotide mutations from the germline of mAbs that react to NANP6 repeat region peptides (circles, n=67) or C-terminal region peptides (squares, n=10) are shown, and indicate that the mAb is from a vaccinee that was protected (green) or not protected (blue) and received a standard (012M, closed symbols) or fractional (Fx017M, open symbols) dose. Figure 1B SPR-determined binding potency (KD) of antibodies (n=141) selected from the 35 most potent lineages tested against CSP and a set of CSP-derived peptides that are homologous to RTS, S (NANP6, NPNA3) or heterologous to RTS, S (NVDP3NANP2, NPDPNANPNVDPNANP, junctions). Examples show an antibody with a broadly promiscuous binding curve (green, AB-007163), an antibody with a relatively biased curve towards homologous peptides (purple, AB-007143) and an antibody with a curve between these extremes (blue, AB-007175). Figures 1C to 1G Linear regressions comparing the number of nucleotide mutations per heavy chain germline (SHM) to the log-transformed SPR binding off-rate (k 解离 ), are shown for Figure 1C short primary repeat (NPNA3, n=140), Figure 1D junction (KQPADGNPDPNANPN, n=68), Figure 1E short secondary repeat (NPDPNANPNVDPNANP, n=109), Figure 1F long primary repeat (NANP6, n=141), and Figure 1G long secondary repeat (NVDP3NANP2, n=129). For correlations of non-transformed data, P<0.03 for all comparisons (Spearman test) and P<0.04 for all comparisons (Pearson test) except P>0.5 for NVDP3NANP2.

[0025] Figures 2A to 2M In vivo pharmacology, SHM and binding of anti-CSP antibodies are shown. Figures 2A to 2C Data from liver burden models are shown, whereFigure 2A Percent inhibition of antibodies (32 lineages) n=69 compared to AB-000317, where colors other than gray represent the six lineages containing the most potent antibodies (*P>0.05, Two-sided, non-parametric log-rank, Figures 2B to 2C Data from AB-000224 and AB-000317 showing luminescence from the parasite in the liver (total flux, photons / sec) Figure 2B ), *P<0.035, and serum concentration of antibody at the time of sporozoite challenge (serum [Ab], pg / ml) Figure 2C ), line and bars represent geometric mean and geometric standard deviation, P>0.2 (“ns”), two-tailed Mann-Whitney test. Figures 2D to 2I Percent inhibition of liver burden inhibition activity normalized to the activity of AB-000317, where each antibody represents significantly better activity than AB-000317 (dark blue triangles), no difference (green circles), or weaker (light blue triangles) with Figures 2D to 2G Binding off-rates (SPR, koff) for CSP (n=70) Figure 2D ), major repeat sequence (NPNA3, n=70) Figure 2E ), junction (KQPADGNPDPNANPN, n=42) Figure 2F ), and minor repeat sequence (NPDPNANPNVDPNANP, n=60) Figure 2G ) peptides, and to Figures 2H to 2I Number of amino acid residue changes from SHM for each antibody (n=70), heavy chain Figure 2H ), and light chain Figure 2I ), showing linear regression of log-transformed data. P<0.005 for all comparisons (Pearson’s test) and P<0.05 for all comparisons (Spearman’s test) except for liver burden inhibition vs. koff[NPDPNANPNVDPNANP] P>0.06 for the correlation of non-transformed data. Figure 2JSPR dissociation (k) and association (kon) rates for NPNA3 peptide binding for antibodies with high SHM (green, >20 mutations per clone, n=56) or low SHM (blue, <20 mutations per clone, n=14) are shown, and activity is weaker (downward triangle), no difference (circle), or better (upward triangle) than AB-000317 (two-sided, non-parametric log-rank). Monoclonals from lineages reported to bind CSP with Fab-Fab homotypic interactions are indicated (red circles, AB-00039942, AB-007159, AB-007160, AB-007161). Figure 2K Risk ratios for n=25 antibodies (14 lineages) compared to AB-000317 in the mosquito bite parasitemia model are shown, where colors other than gray indicate the five lineages containing the most potent antibodies, and Figures 2L to 2M Survival curves for replicate experiments for AB-000224 [0.74 (0.15, 3.8)] ( Figure 2L ) and AB-007088 [0.61 (0.097, 3.8)] ( Figure 2M ) compared to AB-000317, two-sided, non-parametric log-rank [Mantel-Haenszel hazard ratio (95% confidence interval)].

[0026] Figures 3A to 3H CSP-reactive lineages from blood PB after the third dose of RTS,S are illustrated. Figure 3A and Figure 3B IgG lineages per vaccinated (bars, n=45) are shown, which were clonally expanded (i.e., had >2 distinct nucleotide clones; green), were cell expanded but only one IgG clone was observed (i.e., had >2 identical nucleotide clones; gray), or lacked evidence of recent expansion and contained only one observed PB (blue), as Figure 3A , lineage factor or Figure 3B , number of PB per vaccinated is shown. Figure 3C Lineage sizes per vaccinated are shown, calculated by dividing the number of PB in that lineage by the number of PB in all expanded lineages within each group repertoire, and then assigning rank size. Boxes indicate interquartile range, lines within boxes are medians, and thin lines indicate minimum and maximum values per vaccinated for each rank size, where the first four rank sizes contain 33% of the PB in all expanded lineages (dashed line). Figures 3D to 3G ELISA reactivity, SHM level, and vaccinated protection status (n=349) of mAbs from expanded lineages. Figure 3DThe number of nucleotide mutations of the germline (SHM) of mAbs that were non- reactive (dark blue, n=185), showed uncertainty, weak signal (orange, n=29) or reactive (light green, n=135) in CSP ELISA is shown. Domain specificity of CSP reactive mAbs is shown in light green boxes. Monoclonal antibodies were reactive by ELISA to NANP6 repeat region peptide (light green, n=98), C-terminal region peptide (Pfs16, light blue, n=20) or non-reactive in either peptide ELISA (light green, n=9), line is median, ***P<0.0001, **P<0.001, unpaired two-tailed Mann-Whitney test. CSP reactive mAbs not tested in peptide ELISA (n=8) are not shown. Figures 3E to 3G The percentage of tested antibodies that were CSP reactive (82 / 249 and 53 / 100 mAbs respectively; Figure 3E ), repeat region, NANP6 peptide reactive (59 / 244 and 39 / 97 mAbs respectively; Figure 3F ), and subset of only major class sizes 1 to 4 from CSP reactive (52 / 142 and 31 / 46 mAbs respectively; Figure 3G ) are shown from expanded lineages of vaccinated (green, n=36) and unvaccinated (blue, n=9) subjects, **P<0.001, *P<0.01, Fisher’s exact test. In Figure 3H , each symbol represents a lineage for the vaccinated subject shown on the X axis. Lineages from which clones were selected for testing (n=369) are represented by CSP reactivity: CSP reactive (green dots, n=139), uncertainty (orange dots, n=29) or non-reactive (blue triangles, n=201). All non-tested lineages are shown (grey circles, n=13,134; 2,313 expanded lineages and 10,821 single PB lineages). Vaccinated subjects that were protected had a lower ratio of CSP reactive to non-reactive lineages than unvaccinated subjects (bootstrap analysis, P=0.0011). Red circles indicate two lineages containing the amino acid sequence of AB-000317.

[0027] Figure 4 Histograms showing the number of nucleotide mutations of the germline (SHM) of IgG heavy and light chain combinations of PB collected 7 days after administration of a third dose (blue, n=22,319) or fourth dose (grey, n=10,429) of RTS,S are shown.

[0028] Figures 5A to 5I IgG sequences and repertoire features of PB responses after a third dose of RTS,S are shown. Figures 5A to 5CUsage of germline V genes for heavy chains ( Figure 5A ) and light chains ( Figure 5B ) is shown for comparison between protected (green, n = 36) and unprotected (blue, n = 9) subjects and across dose groups (standard dose group, “012M”, n = 15; fractional dose group, “Fx017M”, n = 30), where IGHV3-30, IGHV3-33, KV1-5, KV3-20, and LV1-40 exhibit high prevalence. Figure 5C Specific pairings of heavy and light chain genes are shown. Figure 5D 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 were not associated after correction for multiple hypothesis testing. All P > 0.05, Benjamini-Hochberg or Bonferroni test. No significant association was detected between vaccinee protection status and dose group for IgG heavy chain and light chain constant region subclasses of Figures 5E to 5F Figure 5G Figure 5H Figure 5I No significant association was detected between vaccinee protection status and dose group for repertoire clonality (normalized Shannon entropy) of the analysis including lineages containing only one PB (P > 0.05 for all analyses, Wilcoxon rank-sum test or Kolmogorov-Smirnov test), and length of the complementary determining region 3 (CDR3) of the heavy chain ( Figure 5H ) or light chain ( Figure 5I ). Boxes represent the interquartile range, the line within the box is the median, thin lines represent the most extreme data points within 1.5 times the interquartile range, and points outside the thin lines are plotted as outliers individually.

[0029] Figures 6A to 6C Antibody lineages tested in the binding assay and reactivity to CSP or HBsAg are shown. Rank size of expanded lineages in the repertoire of each protected and unprotected vaccinee group for PB collected 7 days after the third dose of RTS,S. Figure 6AThe expanded PB antibody lineages for each tier size and vaccinee tested in the CSP ELISA (circles indicate >1 lineage) are shown, with the size of the circle being proportional to the fraction of lineages tested among all lineages observed for each tier size and vaccinee. Lineages from vaccinees with the same number of PBs have the same tier size. The largest circle indicates that all lineages from that tier size of vaccinee were tested. The smallest circle indicates that only 1 out of 58 lineages observed from that tier size of vaccinee were tested. In some cases, lineages from vaccinees of a given tier size were not tested (grey bars). Figure 6B and Figure 6C The expanded PB antibody lineages for each tier size and vaccinee tested in the CSP ELISA (circles indicate >1 lineage) are shown, with the size of the circle being proportional to the fraction of lineages tested among all lineages observed for each tier size and vaccinee. Lineages from vaccinees with the same number of PBs have the same tier size. The largest circle indicates that all lineages from that tier size of vaccinee were tested. The smallest circle indicates that only 1 out of 58 lineages observed from that tier size of vaccinee were tested. In some cases, lineages from vaccinees of a given tier size were not tested (grey bars). Figure 6B , CSP ELISA, reactivity (green, n=135 mAbs, 94 circles), uncertainty (grey, n=29 mAbs, 14 circles), no reactivity (blue, n=185 mAbs, 144 circles), or combinations of these results for different mAbs from the same tier size and vaccinee (pie charts of mixed colors, 30 circles) tested, and in Figure 6C , HBsAg ELISA, reactivity (green, n=38 mAbs, 36 circles), uncertainty (grey, n=3 mAbs, three circles), no reactivity (blue, n=77 mAbs, 72 circles), or combinations of these results for different mAbs from the same tier size and vaccinee (pie charts of mixed colors, two circles) tested mAbs for each tier size and vaccinee of the expanded PB antibody lineages (circles indicate 1 to 5 lineages).

[0030] Figure 7A and Figure 7B The SHM and CSP peptide binding of mAbs to RTS,S dose groups and the protection status of vaccinees are shown. The SHM level distribution of mAbs from expanded lineages of vaccinees, Figure 7A , RTS,S administered as a third standard dose (0 12M, dark blue, n=15) or a delayed split dose (Fx 017M, orange, n=30), where mAbs showed reactivity to the CSP repeat region (NANP6, n=45 and n=53, respectively), the CSP C-terminal region (C-term, n=4 and n=16, respectively), or no reactivity to both peptides (negative, n=5 and n=4, respectively) compared to mAbs with no reactivity in the CSP ELISA (n=72 and n=113, respectively), and, Figure 7B, from either protected (green, n=36) or unprotected (light blue, n=9) vaccinees, where mAbs showed reactivity to CSP repeat region (n=59 and n=39, respectively)], to CSP C-terminal region (n=12 and n=8, respectively), or to neither peptide (n=6 and n=3, respectively, negative), compared to mAbs that were non-reactive in the CSP ELISA (n=147 and n=38, respectively), line is median, ***P<0.0001, **P<0.001, *P<0.02 or P>0.1 ("ns"), unpaired two-tailed Mann-Whitney test. DETAILED DESCRIPTION

[0031] DEFINITIONS

[0032] For purposes of interpreting this specification, the following definitions will apply and, where appropriate and applicable, the singular form of a word includes the plural and vice versa. If any definition listed below conflicts with the context of the application, the definition below shall control.

[0033] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th Ed., John Wiley & Sons (New York, N.Y. 1992), provide one of skill with a general guide to many of the terms used in this application.

[0034] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly 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 mixtures of cells, etc.

[0035] As used herein, the term "about" or "approximately" means amounts that are readily understood by those of ordinary skill in the art to be within the range of the intended meaning, e.g., ±20%, ±10%, or ±5%, all within the intended meaning of the recited value.

[0036] As used herein, the term "antibody" means an isolated or recombinant binding agent that comprises the necessary variable region sequences that specifically bind to an epitope of an antigen. Thus, "antibody" as used herein is any form of antibody or fragment thereof that exhibits the desired biological activity (e.g., binds to a particular target antigen). Thus, it 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, and the like, so long as they exhibit the desired biological activity.

[0037] "Antibody fragments" include 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 antibodies produces two identical antigen binding fragments (called "Fab" fragments, each with a single antigen binding site) and a residual "Fc" fragment (a designation reflecting its ability to crystallize readily). Pepsin treatment yields an F(ab')2 fragment that has two antigen binding sites and is still capable of cross-linking antigen.

[0038] As used herein, the terms "anti-CSP antibody" and "CSP antibody" can be used synonymously and refer to an antibody that binds to the Plasmodium falciparum circumsporozoite (CSP) antigen.

[0039] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and vice versa.

[0040] As used herein, "V region" refers to an antibody variable region domain comprising a fragment of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. A heavy chain V region, VH, is the result of V gene (HV), D gene (HD), and J gene (HJ) rearrangement (known as V(D)J recombination) during B cell differentiation. A light chain V region, VL, is the result of V gene (LV) and J gene rearrangement.

[0041] As used herein, “complementarity determining region (CDR)” refers to the three hypervariable regions (HVRs) in each chain that are interposed between the four “framework” regions established by light and heavy chain variable regions. CDRs are the primary contributors to binding with antigenic epitopes. The CDRs of each chain are referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are further identified by the chain in which the particular CDR is found. Thus, a VH CDR3 (HCDR3) is 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” can be used interchangeably with “HVR” when referring to CDR sequences.

[0042] The amino acid sequences of CDRs and framework regions can be determined using various definitions in the art, such as Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Chothia and 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 an antigen binding site is also described below: 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. January 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., Immunomethods, 1, 126, (1992); and Rees et al., In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996). References to CDRs determined by Kabat numbering are, for example, based on Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institute of Health, Bethesda, MD (1991). Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). CDRs can also be determined using available computer modeling systems, such 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.

[0043] An "Fc region" refers to the constant region of an antibody that does not include the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, the Fc can include the J chain. For IgG, the Fc includes the immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy. It is understood in the art that the boundaries of the Fc region can vary, however, the human IgG heavy chain Fc region is usually defined to include the residue C226 or P230, using the EU index of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.) as numbering. The term "Fc region" can refer to this region alone or in the context of an antibody or antibody fragment. The "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 a change to 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 significantly losing biological function. Such variants can be selected according to general rules known in the art so as to have minimal impact on activity (see, e.g., Bowie et al., Science 247:306-1310, 1990). For example, for IgG4 antibodies, a single amino acid substitution can be introduced (S228P according to Kabat numbering; designated IgG4Pro) 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 includes substitutions that improve the pharmacokinetic properties of the antibody, e.g., increase serum half-life. Non-limiting examples of substitutions of the Fc region can be found in U.S. Patent No. 8,088,376, the contents of which are incorporated by reference in their entirety.

[0044] The term "equilibrium dissociation constant," abbreviated as (KD), refers to the dissociation rate constant (kd, time 1 ) divided by the association rate constant (ka, time -1 M -1 The equilibrium dissociation constant can be measured using any method. Thus, in certain embodiments, the 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 typically less than about 10 nM, as determined by a biosensor system such as a Biacore® using a surface plasmon resonance assay performed at 37 °C. determined by surface plasmon resonance analysis (e.g., Biacore® system). In certain embodiments, an antibody of the present disclosure has a KD of less than 5 x 10 -5 M, less than 10 -5 M, less than 5 x 10 -6 M, less than 10 -6 M, less than 5 x 10 -7 M, less than 10 -7 M, less than 5 x 10 -8 M, less than 10 -8 M, less than 5 x 10 -9 M, less than 10 -9 M, less than 5 x 10 -10 M, less than 10 -10 M, less than 5 x 10 -11M , less than 10 -11 M, less than 5 x 10 -12 M, less than 10 -12 M, less than 5 x 10 -13 M, less than 10 -13 M, less than 5 x 10 -14 M, less than 10 -14 M, less than 5 x 10 -15 M, or less than 10 -15 M, or less than 5 x 10 -5 M, less than 10 -5 M, less than 5 x 10 -6 M, less than 10 -6 M, less than 5 x 10 -7 M, less than 10 -7 M, less than 5 x 10 -8 M, less than 10 -8 M, less than 5 x 10 -9 M, less than 10 -9 M, less than 5 x 10 -10 M, less than 10 -10 M, less than 5 x 10 -11 M, less than 10 -11 M, less than 5 x 10 -12 M, less than 10 -12 M, less than 5 x 10 -13 M, less than 10 -13 M, less than 5 x 10 - 14 M, less than 10 -14 M, less than 5 x 10 -15 M, or less than 10 -15KD of M or less, as measured as a monovalent antibody (such as a monovalent Fab). In certain embodiments, an anti-CSP antibody of the present disclosure has a KD of less than 100 pM, e.g., or less than 75 pM, e.g., in the range of 1 pM to 100 pM, when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37 °C. An anti-CSP antibody of the present disclosure has a KD of greater than 100 pM, e.g., a KD in the range of 100 pM to 1000 pM or 200 pM to 1000 pM, when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37 °C. An anti-CSP antibody of the present disclosure has a KD of greater than 100 pM, e.g., a KD in the range of 100 pM to 1000 pM or 200 pM to 1000 pM, when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37 °C.

[0045] The term “monovalent molecule” as used herein refers to a molecule having one antigen binding site, e.g., a Fab or scFv.

[0046] The term “bivalent molecule” as used herein refers to a molecule having two antigen binding sites. In certain embodiments, a bivalent molecule of the present application is a bivalent antibody or a bivalent fragment thereof. In certain embodiments, a bivalent molecule of the present application is a bivalent antibody. In certain embodiments, a bivalent molecule of the present application 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 are composed of multiple bivalent units (2 and 5, respectively), and thus have a higher valency. This bivalency increases the avidity of the antibody for the antigen.

[0047] The term “monovalent binding” or “monovalently binds to” as used herein refers to the binding of one antigen binding site to its antigen.

[0048] The term “bivalent binding” or “bivalently binds to” as used herein refers to the binding of both antigen binding sites of a bivalent molecule to its antigen. In certain embodiments, both antigen binding sites of a bivalent molecule share the same antigen specificity.

[0049] The term “valency” as used herein refers to the number of different binding sites of an antibody for an antigen. A monovalent antibody includes one binding site for an antigen. A bivalent antibody (e.g., a bivalent IgG antibody) includes two binding sites for the same antigen.

[0050] The term "affinity" as used herein refers to the single or combined strength of binding of one or both arms of an antibody (e.g., an IgG antibody) to a simple or complex antigen expressing one or more epitopes. As defined herein, the term "affinity" does not imply a specific number of valencies between the two binding partners.

[0051] The phrase "specifically (or selectively) binds" to an antigen or target or "specifically (or selectively) immunoreacts with" refers to a binding reaction between an antibody and an antigen or target of interest that occurs only when the antigen or target is present in a particular environment, and does not occur in the absence of the antigen or target.

[0052] The term "identical" or percent "identity," in the context of two or more polypeptide sequences, refers to a specified percentage (e.g., at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) of amino acid residues that are the same or identical between two or more sequences or sub-sequences when compared and aligned for maximum correspondence over a comparison window or designated region (e.g., the length of two sequences being compared). For purposes of determining percent amino acid sequence identity, the comparison window can be established at the level of the amino acid residue, with either or both individual amino acids or nucleotides overlapping as a function of the length of sequences compared. Various methods for alignment of sequences for comparison are known in the art, including but not limited to, the alignment of 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 algorithm disclosed 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, BLAST 2.0 can be used with the default parameters to determine percent sequence identity.

[0053] "Substitution" as used herein means the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

[0054] A "conservative" substitution as used herein refers to a substitution of an amino acid such that the charge, polarity, hydrophilicity (hydrophobic, neutral, or hydrophilic), and / or size of the side chain is maintained. Illustrative sets of amino acids that can be substituted for one another include: (i) positively charged amino acids Lys and Arg; and His at a pH of about 6; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr, and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic hydrophobic amino acids Ala, Val, Leu, and He, (vi) hydrophobic sulfur-containing amino acids Met and Cys, which are less hydrophobic than Val, Leu, and He, (vii) small, polar, uncharged amino acids Ser, Thr, Asp, and Asn, (viii) small, hydrophobic or neutral amino acids Gly, Ala, and Pro; (ix) amide-containing amino acids Asn and Gin; and (xi) beta-branched amino acids Thr, Val, and He. Reference to the charge of an amino acid refers to the charge at a pH of 6 to 7.

[0055] As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to both sense and antisense strands of RNA, cDNA, genomic DNA, and the synthetic forms and mixed polymers described above. In certain embodiments, a polynucleotide refers to a polyribonucleotide, a polydeoxynucleotide, or a modified form of either type of nucleotide, and combinations thereof. These terms also include, but are not limited to, DNA in either single- or double-stranded form. Furthermore, a polynucleotide, e.g., a cDNA or mRNA, can include one or more naturally occurring and modified nucleotides linked together by natural and / or non-natural internucleotide linkages. As will be readily understood by those of skill in the art, nucleic acid molecules can be chemically or biochemically modified, or can contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, amino phosphonates, amino -methvl phosphonates, phosphoramidates, etc.); charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.); pendent moieties (e.g., polypeptides); intercalators (e.g., acridine, psoralen, tetracycline, etc.); chelators; alkylators; and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The above list of modifications is not exhaustive and is intended to include any modification which can be made to the nucleic acid structure. The above terms also are intended to include any topological conformation, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpinned, circular, and padlocked conformations. Unless otherwise indicated, a reference to a nucleic acid sequence encompasses its complement. Thus, a reference to a nucleic acid molecule having a specified sequence is understood to encompass its complementary strand and its complementary sequence. The term also includes codon-optimized nucleic acids encoding the same polypeptide sequence.

[0056] An "isolated" nucleic acid is a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that typically comprise 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.

[0057] An "isolated" nucleic acid is a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that typically comprise 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.

[0058] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. A "vector," as used herein, refers to a recombinant construct in which a nucleic acid sequence of interest is inserted. Certain vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0059] 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 a cell. A host cell can be a recombinant host cell, and includes primary transformed cells and progeny that are descended from the original transformed cell, regardless of the number of transfers.

[0060] The term "variant" of a polypeptide, as used herein, is a polypeptide that differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and / or insertions. In the present application, "variant" of a sequence described in the section "Anti-CSP antibody variants" refers to engineered sequences, and not naturally occurring sequences.

[0061] As used herein, a "recombinant antibody" refers to an antibody in which the exact amino acid sequence of the antibody is not found naturally in the given organism (e.g., an antibody from a mammal). In certain embodiments, the term can refer to an antibody that includes one or more amino acid residues that are not found in a naturally occurring antibody. In certain embodiments, a recombinant antibody can have a CDR that includes an amino acid residue that is not found in a naturally occurring antibody (e.g., an antibody from a mammal). In another exemplary embodiment, a recombinant antibody can have a framework (FR) that includes an amino acid residue that is not found in a naturally occurring antibody (e.g., an antibody from a mammal). In certain embodiments, a recombinant antibody can have a constant region that includes an amino acid residue that is not found in a naturally occurring antibody (e.g., an antibody from a mammal).

[0062] In the context of describing the binding strength of two antibodies to the same target, the term "comparable" refers to two dissociation constant (KD) values calculated from two binding reactions that are within three (3) fold of each other. In certain embodiments, the ratio between a first KD (KD of the binding reaction between the first antibody and the target) and a second KD (KD of the binding reaction between the second antibody and the target) is in the range of 1 :3 or 3: 1, exclusive. A smaller KD value indicates stronger binding. For example, without being bound by any limitation, an antibody variant that has stronger binding compared to AB-000224 binds to the target with a KD that is at least 1 / 3 of the KD measured for AB-000224 against the same target.

[0063] Anti-CSP antibodies

[0064] The present disclosure provides anti-CSP antibodies and variants thereof. The malaria antibodies disclosed herein were discovered in an antibody repertoire generated by the 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. The technology and its use in antibody discovery are well known and disclosed, for example, in WO 2012148497A2, the entire contents of which are incorporated herein by reference. The RTS,S vaccine is a pseudo-virion particle vaccine that combines the hepatitis B surface antigen along with the central repeat region and C-terminal region of 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 to 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):el001685, (2014), WO 1993 / 10152.

[0065] CSP comprises three main domains: i) the N-terminus; ii) the central repeat (CR) region, which consists of multiple (25 to 40) tetrapeptides of NANP (“major repeat sequence”), interspersed with NPDP tetrapeptides and 2 to 4 NVDP (“minor repeat sequence”) tetrapeptides; and iii) the C-terminal domain. The central repeat region of CSP is highly immunogenic, and in all P. falciparum strains for which CSP sequences are available, the repeat region consists of 1 NPDP repeat sequence, 3 to 5 NVDP repeat sequences, and 35 to 41 NANP repeat sequences (e.g., there are a total of 1 / 4 / 38 NPDP / NVDP / NANP motifs in the P. falciparum 3D7 strain). The repeat region begins with a junction NPDP sequence, is usually followed by three alternations of NANP and NVDP sequences, and continues with the remaining NANP repeat sequences, with most P. falciparum strains having one NVDP interspersed among the long NANP repeats. Pholcharee, T. et al., J. Mol. Bio. 432: 1048-1063 (2020).

[0066] Analysis of the anti-CSP antibodies disclosed herein indicates that the percentage of plasmablasts expressing CSP-specific IgG is inversely related to protection among antibodies isolated after the third dose of vaccine (P3D). These data suggest that, despite the well-reported association between anti-CSP antibodies and protection, more anti-CSP-expressing cells, NANP repeat-binding antibodies can not drive greater protection. This result can indicate that the presence of only effective inhibitory antibodies at P3D plasmablasts is insufficient for protection. Rather, the relative levels of such antibodies to other repeat-binding antibodies can be important for providing consistent protection.

[0067] This analysis additionally indicates that the inhibitory activity of antibodies against sporozoites in vivo is not related to the binding kinetics of long NANP6 peptides (Table 9), but is significantly related to the koff of CSP, as well as to the binding kinetics of peptides containing short NANP (NPNA3) and the minor repeat region and junction region (JR) (Table 9). These data suggest that protective antibodies induced after RTS,S vaccination mature to short NANP repeat sequences, but also acquire or retain promiscuous binding activity to minor repeat sequences and junction epitopes that are not present in RTS,S. Consistent with this interpretation, the level of SHM aggregation in the heavy and light chains of protective antibodies is related to the binding kinetics of short peptides containing NANP, short peptides containing NVDP, and short peptides containing NPDP (Table 9), as well as to inhibitory activity in the sporozoite challenge model (Table 9). Thus, affinity maturation and functionality appear to be related to short peptide sequences included and excluded from RTS,S, but not to long NANP repeat sequences. Figures 1C to 1E Figures 2H to 2I ​​

[0068] Furthermore, the correlation between in vivo activity and binding kinetics of NVDP-containing peptides and NPDP-containing peptides not present in RTS,S ( Figures 2F to 2G , Table 9); Correlation between in vivo activity and binding kinetics for the short NPNA3, but not the longer NANP6 peptide ( Figure 2E , Table 9); and the inverse relationship between the reactivity of the NANP6-expanded antibody repertoire and protection against CHMI is consistent with the hypothesis that multiple NANP repeats act as immune "bait" to divert and dilute protective immunity. Under this hypothesis, antibody repertoires that only bind to NANP repeat region epitopes but provide limited protection are preferentially expanded, thereby diluting the protective capacity of the broader anti-CSP repertoire. Promiscuous antibodies that also bind to multiple NANP repeats and are similarly present at high density on CSP can further enhance the antibody association rate to heterologous epitopes. Therefore, the avidity provided by promiscuous binding can drive more protective responses in vivo.

[0069] In certain embodiments, the anti-CSP antibodies disclosed herein bind to a first epitope present in the central repeat region of CSP and bind 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.

[0070] In certain embodiments, in addition to an epitope comprising NPNA, the anti-CSP antibodies disclosed herein also bind to a second epitope that is heterologous to an epitope present in the RTS,S vaccine (referred to herein as a heterologous epitope). Heterologous epitopes include: an epitope of the minor repeat region of CSP that includes an epitope comprising DPNA / NPNV; and an epitope of the joining region of CSP that includes an epitope comprising DPNA.

[0071] Anti-CSP antibodies and their variants

[0072] 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, for example, as shown by a reduction in parasite numbers in a mouse model of malaria infection.

[0073] In certain embodiments, the anti-CSP variants disclosed herein retain the binding specificity, activity, and stability and / or manufacturing properties of the parent antibody. In certain embodiments, the anti-CSP variants disclosed herein have improved developability, e.g., as identified by various in vitro assays such as aggregation assessment by HPLC or UPLC, hydrophobic interaction chromatography (HIC), multispecific assay (e.g., baculovirus particle binding), self-interaction nanoparticle spectroscopy (SINS), or mass spectrometry analysis following incubation under accelerated degradation conditions such as high temperature, low pH, high pH, or oxidative H2O2. A mutation is successful if the activity is retained (or enhanced) while the severity of the susceptibility is removed or reduced.

[0074] Antibody susceptibility is further described in Table 1 below:

[0075] Table 1. Description of potential development susceptibility

[0076]

[0077]

[0078] 1 “Free cysteine” refers to a cysteine that is not forming a disulfide bond with another cysteine, and thus is “free” in the form of a thiol. The presence of a free cysteine in an antibody can be a potential development susceptibility. In general, an odd net number of cysteines in a protein indicates the potential for the presence of a free cysteine.

[0079] 2 An N-linked glycosylation site is N-X-S / T, where X is any residue except proline.

[0080] 3 Sharma et al., Proc. Natl. Acad. Sci. USA 111:18601-18606, 2014.

[0081] 4 The motif consists of a K or R, followed by a K or R. In other words, the motif can be KK, KR, RK, or RR.

[0082] 5 The dipeptide NG causes a moderate risk of development susceptibility. The dipeptides NA, NN, NS, and NT cause a low risk of development susceptibility. N can also exhibit a low susceptibility risk to other succeeding residues (e.g., D, H, or P). In other words, the dipeptides ND, NH, or NP cause a low risk of development susceptibility.

[0083] 6Similar to the above, the dipeptide DG poses a moderate risk of development susceptibility. The dipeptides DA, DD, DS, and DT pose a low risk of development susceptibility. D can also exhibit a low risk of development susceptibility to other succeeding residues (e.g., N, H, or P).

[0084] Another goal of engineering variants is to reduce the risk of clinical immunogenicity. For example, to reduce the generation of anti-drug antibodies against the therapeutic antibody. In certain embodiments, the anti-CSP antibody variants have reduced immunogenicity compared to the parent antibody.

[0085] Factors that drive clinical immunogenicity can be divided into two groups. First are factors inherent to the drug, such as sequence, post-translational modifications, aggregates, degradation products, and contaminants. Second are factors related to the way the drug is used, such as dose level, frequency of dosing, route of administration, patient immune status, and patient HLA type.

[0086] One approach to engineering variants to be as self-like as possible is to identify proximal germline sequences and mutate as many of the mismatched positions (also called “germline deviations”) as possible to the germline residue type. This approach is applicable to the germline genes IGHV, IGHJ, IGKV, IGKJ, IGLV, and IGLJ, and considers all variable heavy (VH) and variable light (VL) regions except for a portion of H-CDR3. The germline gene IGHD encodes a portion of the H-CDR3 region, but generally exhibits too much variation (e.g., forward or reverse orientation, any of three reading frames, and 5’ and 3’ modifications and non-templated additions) in the way it recombines with IGHV and IGHJ to present a “self” sequence template from a population perspective.

[0087] Each germline gene can exhibit as different alleles in a population. In terms of minimizing the percentage of patients with an immunogenic response, the candidate drug with the lowest immunogenicity can be the candidate drug that matches the alleles that are common 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.

[0088] Another approach to engineering reduced immunogenicity risk is to use in silico predictions of immunogenicity, such as predictions of T cell epitopes, or in vitro assays of immunogenicity, such as ex vivo human T cell activation. For example, services such as offered by Lonza in the United Kingdom employ a platform to predict HLA binding and in vitro assessment to further identify potential epitopes.

[0089] In certain embodiments, antibody variants are additionally designed to enhance the efficacy of the antibody. Design parameters for this aspect focus on CDRs (e.g., CDR3). Positions to mutate are identified based on structural analysis of antibody-antigen co-crystals (Oyen et al., Proc. Natl. Acad Sci. USA 114:E10438-E10445, 2017) and sequence information of other antibodies of the same lineage as AB-000224 or AB-007088.

[0090] Mutation design methods

[0091] Development susceptibility can be removed or reduced by one or more mutations. Mutations are designed to preserve antibody structure and function while removing or reducing development susceptibility and improving function. In certain embodiments, mutations of chemically similar residues are identified to maintain size, shape, charge, and / or polarity. Non-limiting examples of mutations are described in Table 2 below:

[0092] Table 2

[0093]

[0094] In certain embodiments, variants of the anti-CSP antibodies disclosed herein comprise modifications as compared to the parent antibody that provide improved pharmacokinetic properties, increased serum stability, stronger binding, and / or improved in vivo protection as compared to the parent. In certain embodiments, variants of the anti-CSP antibodies disclosed herein exhibit reduced immunogenicity and / or increased manufacturability as compared to the parent. In certain embodiments, variants of the 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, and have improved developability, e.g., reduced heterogeneity, increased yield, increased stability, improved net charge to improve pharmacokinetics, and / or reduced immunogenicity. In certain embodiments, the VH region or VL region of such variants of the anti-CSP antibodies disclosed herein have at least two, three, four, five, or six or more modifications, e.g., substitutions.

[0095] In certain embodiments, a variant of an anti-CSP antibody disclosed herein exhibits increased serum half-life compared to the parent antibody. In certain embodiments, a variant of an anti-CSP antibody disclosed herein has at least one modification, e.g., substitution, relative to the native Fc region of the heavy chain or light chain sequence described herein, and has improved pharmacokinetic properties, e.g., half-life. In certain embodiments, the Fc region of the heavy chain or the Fc region of the light chain of such a variant of an anti-CSP antibody disclosed herein has at least two, three, four, five, or six or more modifications, e.g., substitutions. In certain embodiments, a variant of an anti-CSP antibody disclosed herein collectively has 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 heavy chain and light chain) 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 include 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, wherein the numbering is defined by the EU index as in Kabat. In certain embodiments, the Fc region of the heavy chain of a variant of an anti-CSP antibody disclosed herein includes a leucine at position 428 and a serine at position 434, wherein the numbering is defined by the EU index as in Kabat.

[0096] Antibody sequences and variants thereof

[0097] In certain embodiments, the anti-CSP antibodies disclosed herein include 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%, about 99% identical to an amino acid sequence set forth in SEQ ID Nos. 1-461, as shown 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%, about 99% identical to an amino acid sequence set forth in SEQ ID Nos. 462-922, as shown 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%, about 99% identical to an 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%, about 99% identical to an amino acid sequence set forth in SEQ ID Nos. 462-922.

[0098] In certain embodiments, the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID Nos. 1-461, as shown in Table 3. In certain embodiments, the light chain variable region comprises an amino acid sequence set forth in SEQ ID Nos. 462-922, as shown in Table 3. In certain embodiments, a) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID Nos. 1-461; and b) the light chain variable region comprises an amino acid sequence set forth in SEQ ID Nos. 462-922.

[0099] In certain embodiments, the heavy chain variable region consists of an amino acid sequence set forth in SEQ ID Nos. 1-461, as shown in Table 3. In certain embodiments, the light chain variable region consists of an amino acid sequence set forth in SEQ ID Nos. 462-922, as shown in Table 3. In certain embodiments, a) the heavy chain variable region consists of an amino acid sequence set forth in SEQ ID Nos. 1-461; and b) the light chain variable region consists of an amino acid sequence set forth in SEQ ID Nos. 462-922.

[0100] In certain embodiments, the heavy chain variable region comprises the CDR1, CDR2, and CDR3 of the heavy chain variable sequences set forth in SEQ ID NOs: 1 to 461, as set forth in Table 3. In certain embodiments, the light chain variable region comprises the CDR1, CDR2, and CDR3 of the light chain variable sequences set forth in SEQ ID NOs: 462 to 922, as set forth in Table 3. In certain embodiments, a) the heavy chain variable region comprises the 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 the CDR1, CDR2, and CDR3 of the light chain variable sequences set forth in SEQ ID NOs: 462 to 922.

[0101] In certain embodiments, the anti-CSP antibodies disclosed herein comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 and having an amino acid sequence set forth in SEQ ID NOs: 1 to 461, as shown in Table 3. In certain embodiments, the anti-CSP antibody variants comprise a light chain variable region comprising CDR1, CDR2, and CDR3 and having an amino acid sequence set forth in SEQ ID NOs: 462 to 922, as shown in Table 3. In certain embodiments, the anti-CSP antibody variants comprise: a) a heavy chain variable region comprising CDR1, CDR2, and CDR3 and having an amino acid sequence set forth in SEQ ID NOs: 1 to 461; and b) a light chain variable region comprising CDR1, CDR2, and CDR3 and having an amino acid sequence set forth in SEQ ID NOs: 462 to 922. Table 3 is provided below.

[0102] In certain embodiments, the anti-CSP antibody variant is designated as shown in Table 3. For example, but not limited to, the anti-CSP antibody variant is designated as "AB-001558", which comprises: 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.

[0103] Table 3

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190] Epitope

[0191] In certain embodiments, the anti-CSP antibodies disclosed herein bind to a first epitope of CSP. CSP is composed of an N-terminal domain containing heparan sulfate binding sites for hepatocyte adhesion, a central repeat region, and a structured C-terminal alpha-thrombospondin repeat sequence (aTSR) that is followed by a GPI anchor that attaches CSP to the sporozoite membrane. The central repeat region of CSP is highly immunogenic and, in all P. falciparum strains with available CSP sequences, the repeat region consists of 1 NPDP repeat, 3-5 NVDP repeats, and 35-41 NANP repeats (e.g., there are a total of 1 / 4 / 38 NPDP / NVDP / NANP motifs in the P. falciparum 3D7 strain). The repeat region begins with the NPDP sequence, is usually followed by three alternations of NANP and NVDP sequences, and continues with the remaining NANP repeats, with most P. falciparum strains having one NVDP interspersed among the long NANP repeat region. Pholcharee, T. et al., J. Mol. Bio. 132: 1048-1063 (2020).

[0192] 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 P. falciparum CSP protein in the repeat region and / or junction region containing 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.

[0193] In certain embodiments, the first epitope comprises the amino acid sequence set forth in SEQ ID NO: 923-974. In certain embodiments, the first epitope consists of the amino acid sequence set forth in SEQ ID NO: 923-974. SEQ ID NOs: 923-974 are provided in Table 4 below.

[0194] Table 4.

[0195] Sequence SEQ ID Sequence SEQ ID NPNA 923 NPNANPNANP 949 NANP 924 PNANPNANPN 950 ANPN 925 NANPNANPNAN 951 PNAN 926 ANPNANPNANP 952 NANPN 927 NPNANPNANPN 953 ANPNA 928 PNANPNANPNA 954 NPNAN 929 NANPNANPNANP 955 PNANP 930 ANPNANPNANPN 956 NANPNA 931 NPNANPNANPNA 957 ANPNAN 932 PNANPNANPNAN 958 NPNANP 933 NANPNANPNANPN 959 PNANPN 934 ANPNANPNANPNA 960 NANPNAN 935 NPNANPNANPNAN 961 ANPNANP 936 PNANPNANPNANP 962 NPNANPN 937 NANPNANPNANPNA 963 PNANPNA 938 ANPNANPNANPNAN 964 NANPNANP 939 NPNANPNANPNANP 965 ANPNANPN 940 PNANPNANPNANPN 966 NPNANPNA 941 NANPNANPNANPNAN 967 PNANPNAN 942 ANPNANPNANPNANP 968 NANPNANPN 943 NPNANPNANPNANPN 969 ANPNANPNA 944 PNANPNANPNANPNA 970 NPNANPNAN 945 NANPNANPNANPNANP 971 PNANPNANP 946 ANPNANPNANPNANPN 972 NANPNANPNA 947 NPNANPNANPNANPNA 973 ANPNANPNAN 948 PNANPNANPNANPNAN 974

[0196] 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 kind of pseudo-virion particle vaccine that combines the hepatitis B surface antigen as well as the central repetitive region and C-terminal region of Plasmodium falciparum (P. falciparum) circumsporozoite protein (CSP). RTS,S consists of two polypeptides; RTS is a single polypeptide chain corresponding to amino acids 207 to 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.

[0197] In certain embodiments, the second epitope comprises a minor repeat region or a junction region of CSP. In certain embodiments, the second epitope comprises a minor repeat region and a junction region of CSP. In certain embodiments, the minor repeat sequence is a minor repeat sequence containing DPNA / NPNV. In certain embodiments, the junction region is a junction region containing DPNA / NPNV. In certain embodiments, the second epitope comprises an amino acid sequence set forth in SEQ ID NO: 975 to 1195. In certain embodiments, the second epitope consists of an amino acid sequence set forth in SEQ ID NO: 975 to 1195. SEQ ID NO: 975 to 1195 are provided in Table 5 below.

[0198] Table 5.

[0199]

[0200]

[0201]

[0202] 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 an amino acid sequence set forth in SEQ ID NO. 923-974 and the second epitope comprises an amino acid sequence set forth in SEQ ID NO. 975-1195. In certain embodiments, the first epitope consists of an amino acid sequence set forth in SEQ ID NO. 923-974 and the second epitope consists of an amino acid sequence set forth in SEQ ID NO. 975-1195. In certain embodiments, the first epitope consists of an amino acid sequence set forth in SEQ ID NO. 923-974, the second epitope consists of an amino acid sequence set forth in SEQ ID NO. 975-1195, and the at least one additional epitope consists of an amino acid sequence set forth in SEQ ID NO. 975-1195.

[0203] Glycosylation of Anti-CSP Antibodies and Variants Thereof

[0204] 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 typically post-translationally modified via the addition of N-glycans at specific asparagine residues on the antibody heavy chain. IgG molecules bear an N-linked glycosylated asparagine per heavy chain. It has been shown that modified glycosylation profiles can modulate antibody function. For example, without being bound by any limitation, altered glycosylation can improve the binding affinity or half-life of an antibody compared to unmodified forms.

[0205] In certain embodiments, the present disclosure provides anti-CSP antibodies and variants thereof having modified glycosylation. In certain embodiments, the antibodies disclosed herein include Fc regions with increased glycosylation. In certain non-limiting embodiments, the Fc regions with increased glycosylation include increased amounts of bisected oligosaccharides. In certain embodiments, the Fc regions with increased glycosylation include increased amounts of non-fucosylated oligosaccharides. In certain embodiments, the Fc regions with increased glycosylation include increased amounts of fucose-containing oligosaccharides.

[0206] In certain embodiments, the antibodies disclosed herein include Fc regions with decreased glycosylation. In certain non-limiting embodiments, the Fc regions with decreased glycosylation include decreased amounts of bisected oligosaccharides. In certain embodiments, the Fc regions with decreased glycosylation include decreased amounts of non-fucosylated oligosaccharides. In certain embodiments, the Fc regions with increased glycosylation include decreased amounts of fucose-containing oligosaccharides.

[0207] In certain embodiments, the antibodies disclosed herein include a V-region with increased glycosylation. In certain non-limiting embodiments, the V-region with increased glycosylation includes an increased amount of bisected oligosaccharides. In certain embodiments, the V-region with increased glycosylation includes an increased amount of non-fucosylated oligosaccharides. In certain embodiments, the V-region with increased glycosylation includes an increased amount of fucose-containing oligosaccharides.

[0208] In certain embodiments, the antibodies disclosed herein include a V-region with reduced glycosylation. In certain non-limiting embodiments, the V-region with reduced glycosylation includes a reduced amount of bisected oligosaccharides. In certain embodiments, the V-region with reduced glycosylation includes a reduced amount of non-fucosylated oligosaccharides. In certain embodiments, the V-region with increased glycosylation includes a reduced amount of fucose-containing oligosaccharides.

[0209] In certain embodiments, modified glycosylation can be obtained by expressing any of the antibodies disclosed herein in a host cell having an altered glycosylation machinery. For example, without being limited in any way, a host cell can include a functional disruption of a fucosyltransferase gene and an antibody expressed in that host cell, wherein reduced glycosylation, e.g., reduced fucosylation, is shown (see PCT Patent Publication No. WO 99 / 54342).

[0210] 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 altered glycosylation acceptor site. In certain embodiments, the alteration includes an elimination of a glycosylation acceptor site. In certain embodiments, the alteration includes a modification of a glycosylation acceptor site. In certain embodiments, the alteration includes an insertion of a glycosylation acceptor site.

[0211] As used herein, a "glycosylation acceptor site" refers to an amino acid residue of a light chain or a heavy chain of an antibody that can be N-glycosylated 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 residue, a threonine residue, a tyrosine residue, a hydroxylysine residue, or a hydroxyproline residue.

[0212] In certain embodiments, the Fc region of an antibody disclosed herein includes one or more glycosylation acceptor sites. In certain embodiments, the V-region of any of the antibodies disclosed herein includes one or more glycosylation acceptor sites. In certain embodiments, the light chain of any of the antibodies disclosed herein includes one or more glycosylation acceptor sites. In certain embodiments, the heavy chain of any of the antibodies disclosed herein includes one or more glycosylation acceptor sites. In certain embodiments, the light chain variable region of any of the antibodies disclosed herein includes one or more glycosylation acceptor sites. In certain embodiments, the heavy chain variable region of any of the antibodies disclosed herein includes one or more glycosylation acceptor sites.

[0213] PEGylation and other chemical modifications of anti-CSP antibodies and variants thereof

[0214] The present disclosure provides anti-CSP antibodies and variants thereof comprising additional modifications. In certain embodiments, the modifications can improve the pharmacological properties of the antibodies, such as half-life. In certain non-limiting embodiments, the modifications include PEGylation, deamination, polymer derivatization, lipidation, removal and / or introduction of disulfide bonds, oxidation, and removal of C-terminal lysine

[0215] In certain embodiments, the modification is PEGylation. PEGylation of antibodies and engineered antibodies includes the attachment of one or more polyethylene glycol (PEG) to the antibody. For example, in certain non-limiting embodiments, PEGylation can be performed by acylation or alkylation reactions with reactive PEG molecules (or similar reactive water-soluble polymers). 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 to C10) alkoxy- or aryloxy- polyethylene glycols or polyethylene glycol-maleimide.

[0216] In certain embodiments, the modification is derivatization with a hydrophilic polymer. For example, in certain non-limiting embodiments, the hydrophilic polymer can be carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene / maleic acid copolymer, polyamino acids (homopolymers or random copolymers) and dextran or poly( n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxy ethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0217] In certain embodiments, the modification is lipidation. Lipidation is the conjugation of a protein with a lipid. Lipidation of peptides improves metabolic stability, membrane permeability, bioavailability, and alters the pharmacokinetic and pharmacodynamic properties of the peptide. For example, lipidated peptides have high affinity for serum albumin, increasing half-life and stability. For example, in certain non-limiting embodiments, the lipid can be myristic acid, palmitic acid, stearic acid, lauric acid, cholesterol, and mixtures thereof.

[0218] 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. In certain redox conditions, two cysteines can form a non-native disulfide bond. In certain non-limiting embodiments, the disulfide bond improves the stability of the antibody, e.g., correct pairing of antibody chains. In certain embodiments, the cysteine is introduced in the V region. In certain embodiments, the cysteine is introduced in the Fc region. In certain embodiments, the modification is a substitution of an amino acid residue to remove a disulfide bond. In certain embodiments, the amino acid substitution removes a cysteine. In certain embodiments, the cysteine is replaced with a serine. In certain non-limiting embodiments, the removal of the cysteine improves the stability of the antibody, e.g., 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.

[0219] Anti-CSP antibody and anti-CSP antibody variant conjugates

[0220] In certain embodiments, the present disclosure provides an anti-CSP antibody or variant thereof conjugated or linked to a therapeutic and / or imaging / detectable moiety. For example, without being limited in any way, the anti-CSP antibody or variant thereof can be conjugated to a detectable marker, a toxin, or a therapeutic agent. The moiety can be linked to the antibody covalently or through a non-covalent bond.

[0221] 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 a cytotoxic agent including, but not limited to, ricin A chain, doxorubicin, daunorubicin, maytansine, taxol, ethidium bromide, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicin, dihydroxy anthracin dione, actinomycin, diphtheria toxin, exotoxin A from Pseudomonas, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, calicheamicin, gelonin, mitogellin, restrictocin, cobra venom factor, ribonuclease, phenomycin, enomycin, curicin, phytolaccin, a calcyphosine, an auristatin, a duocarmycin, a dolastatin, cc1065, or cisplatin. In certain embodiments, the antibody or variant thereof can be linked to an agent such as an enzyme inhibitor, a proliferation inhibitor, a lytic agent, a DNA or RNA synthesis inhibitor, a membrane permeability modulator, a DNA metabolite, a dichloromethyl sulfide derivative, a protein production inhibitor, a ribosome inhibitor, or an apoptosis inducer.

[0222] In certain embodiments, the antibody or variant thereof can be linked to a radionuclide, iron-related compound, dye, fluorescent agent, or imaging agent. In certain embodiments, the antibody can be linked to an agent such as, but not limited to, a metal; a metal chelator; a lanthanide; a lanthanide chelator; a radioactive metal; a radioactive metal chelator; a positron emitting nucleus; a microbubble (for use in ultrasound); a liposome; a molecule microencapsulated in a liposome or nanosphere; a single crystal 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.

[0223] In certain embodiments, the present disclosure provides bispecific molecules comprising an anti-CSP antibody, variant thereof, or fragment thereof disclosed herein. The anti-CSP antibody, anti-CSP antibody variant, or antigen-binding portion thereof can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or receptor ligand), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. An anti-CSP antibody or variant thereof disclosed herein can be derivatized or linked to more than one other functional molecule to generate a multispecific molecule that binds to more than two 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 generate a bispecific molecule of the present application, an antibody of the present application can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent associations, or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, to generate a bispecific molecule. In certain non-limiting embodiments, for example and without limitation, a knob-in-hole strategy can be used to generate bispecific antibodies. This strategy generally involves generating a front half of a first antibody that recognizes a first antigen (e.g., CSP) and a back half of an antibody that recognizes a second antigen or binding site, and then linking the two halves together to generate a bispecific antibody. In certain embodiments, the first antigen and the second antigen are different epitopes of a CSP protein.

[0224] Activity

[0225] The activity of any of the anti-CSP antibodies disclosed herein can be assessed by using different endpoints. In certain embodiments, the activity of binding to CSP (to a series of linear peptides of varying lengths representing the immunodominant regions of the CSP protein, or to the entire CSP protein) is assessed. In certain embodiments, the ability to protect against challenge by a Plasmodium (including P. falciparum CSP), e.g., in an in vivo malaria animal model, is assessed. In certain embodiments, effector function, e.g., ADCC, is also assessed.

[0226] 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. For example, and without being limited in any way, systems suitable for SPR use are LSATM (Carterra, Dublin, CA), BiacoreTM (General Electric, Boston, MA), and OpenSPR (Nicoya, East Kitchener, ON, Canada). In an exemplary SPR assay, each antibody can be directly immobilized to a Carterra CMD200M chip, or can be captured to a CMD200M Carterra chip with a goat anti-human IgG Fc antibody. Unconjugated antibodies can be washed away, and various concentration gradients of the target can be flowed over the antibodies. In certain experimental conditions, the highest concentration of each target can be in the range of 0.5 to 8 pg / mL. To improve accuracy, each antibody can be immobilized at different locations on the chip (e.g., at least 2), and multiple (e.g., 4 to 5) target concentrations can be used to determine the affinity of each antibody-target combination according to standard methods. Antibody-target measurements are repeated if the difference between two replicates is >3-fold.

[0227] In certain embodiments, the binding activity of the anti-CSP antibodies disclosed herein to P. falciparum CSP protein can be assessed by bio-layer interferometry (BLI). For BLI, each of the antigens can be immobilized on a sensor according to the manufacturer’s instructions. Systems suitable for BLI use include, but are not limited to, OctetTM (ForteBio, Fremont, CA) and GatorTM (Probelife, Palo Alto, CA). In certain embodiments, for example and without being limited in any way, the antigens can be biotinylated and immobilized to a streptavidin sensor. To improve accuracy, each antibody can be evaluated in duplicate at a suitable concentration (e.g., 5 pg / mL). Antibody-target measurements are repeated if the difference between two replicates is >3-fold. Assays are typically performed under conditions according to the manufacturer’s instructions. Assays can be performed at temperatures ranging from 20 °C to 37 °C, for example from 20 °C to 25 °C. In certain embodiments, assays are performed at 25 °C. In certain embodiments, assays are performed at 37 °C.

[0228] In certain embodiments, binding to CSP protein is assessed in a competitive assay format with reference antibody A. In certain embodiments, the variant anti-CSP antibodies disclosed herein can block the binding of the reference antibody in the competition assay by about 50% or more.

[0229] The anti-CSP antibodies and anti-CSP antibody variants of the disclosure can also be evaluated for their ability to mediate FcR-dependent activity in various assays.

[0230] In certain embodiments, activity of an anti-CSP antibody can be assessed in vivo in an animal model, e.g., as described in the Examples section. For example, in certain non-limiting embodiments, a mouse malaria liver burden assay can be used, e.g., 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. For example, parasite liver burden can be assessed by RT-qPCR or bioluminescence using an IVIS Spectrum imager. Reduction in parasite liver burden reflects prophylactic activity of the antibody.

[0231] In certain embodiments, activity of an anti-CSP antibody can be determined by assessing in vivo protection and survival of an animal model, e.g., a mouse. For example, but not by way of any limitation, mice are administered the antibody and challenged with transgenic P. berghei expressing P. falciparum CSP protein. In vivo protection can be determined by detecting blood stage parasitemia in a microscope. Survival can be determined by the absence of parasitemia over an observation period, e.g., two weeks immediately following challenge. An increase in survival reflects prophylactic and / or therapeutic activity of the antibody.

[0232] In certain embodiments, an anti-CSP antibody disclosed herein has 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 assessed under the same assay conditions. In certain embodiments, an anti-CSP antibody exhibits improved activity, i.e., greater than 100% activity, compared to antibody AB-000317. In certain non-limiting embodiments, an anti-CSP antibody disclosed herein exhibits 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, an anti-CSP antibody disclosed herein exhibits at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60% or 70% or more increase in survival compared to antibody AB-000317.

[0233] 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 assessed under the same assay conditions. In certain embodiments, the anti-CSP antibodies exhibit improved activity, i.e., greater than 100% activity, compared to AB-000224. 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 assessed under the same assay conditions. In certain embodiments, the anti-CSP antibodies exhibit improved activity, i.e., greater than 100% activity, compared to AB-007088. 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 the activities measured are within 30%, within 25%, within 20%, within 15%, within 10%, within 8%, or within 5% of each other.

[0234] Generation of antibodies

[0235] The CSP antibodies and variants thereof disclosed herein can be produced using vectors and recombinant methods (see, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Ausubel, Current Protocols in Molecular Biology). Reagents for genetic manipulation, cloning vectors, and kits are available from commercial suppliers.

[0236] The present disclosure provides isolated nucleic acids encoding the VH region and / or VL region of any of the anti-CSP antibodies and anti-CSP antibody variants disclosed herein or fragments thereof. In certain embodiments, the present disclosure provides vectors comprising the nucleic acids and host cells into which the nucleic acids are introduced, the host cells being used to replicate the nucleic acids encoding the antibodies and / or express the antibodies. These nucleic acids can encode an amino acid sequence comprising the VL of an anti-CSP antibody or variant thereof and / or an amino acid sequence comprising the VH of an anti-CSP antibody or variant thereof (e.g., the light chain and / or the heavy chain of an antibody). In certain embodiments, the host cells contain: (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.

[0237] In certain embodiments, the present disclosure provides a method of making an anti-CSP antibody disclosed herein. In certain embodiments, the method comprises culturing a previously described host cell under conditions suitable for expression of the antibody. In certain embodiments, the antibody is then recovered from the host cell (or host cell culture medium).

[0238] Suitable vectors containing a polynucleotide encoding an antibody of the present disclosure or fragment thereof include cloning vectors and expression vectors. While the cloning vector selected can vary depending on the intended use of the host cell, useful cloning vectors can generally self- replicate, can have a single target for a particular restriction endonuclease, and / or can carry a gene that confers a selectable marker that can be used in the selection of 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, Col El plasmids, pCR1, RP4, phage DNA, and shuttle vectors.

[0239] Expression vectors are generally replicable polynucleotide constructs containing a nucleic acid of the present disclosure. Expression vectors can replicate in host cells either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids and viral vectors, including adenovirus, adeno-associated virus, retrovirus, and any other vector.

[0240] Suitable host cells for expressing an anti-CSP antibody or anti-CSP antibody variant disclosed herein include prokaryotic or eukaryotic cells. For example, but not by way of any limitation, an anti-CSP antibody can be produced in bacteria, particularly when glycosylation and Fc effector functions are not needed. Following expression, the antibody can be isolated from the bacterial cell lysate in a soluble fraction and can be further purified. Alternatively, the host cell can be a eukaryotic host cell, including, but not limited to, eukaryotic microorganisms such as filamentous fungi or yeast, fungal and yeast strains whose glycosylation pathway has been “humanized” to produce antibodies with partial or fully human glycosylation patterns, vertebrate, invertebrate, and plant cells. Non-limiting examples of invertebrate cells include insect cells. A number of baculoviral strains have been identified that can be used in conjunction with insect cells. Plant cell cultures can also be utilized as the host cells.

[0241] In certain embodiments, vertebrate host cells are used to produce the anti-CSP antibodies of the present disclosure. For example, without limitation, mammalian cell lines that can be used for expression of anti-CSP antibodies include monkey kidney CV1 line transformed by SV40 (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 (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. In certain embodiments, mammalian cell lines that can be used for expression of anti-CSP antibodies can be Chinese hamster ovary (CHO) cell lines; DHFR- CHO cell lines (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); and myeloma cell lines such as YO, NSO, and Sp2 / 0. Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells.

[0242] Host cells transfected with expression vectors or fragments thereof encoding the anti-CSP antibodies of the present disclosure can be cultured under appropriate conditions to allow expression of the polypeptides to occur. The polypeptides can be secreted and isolated from the mixture of cells and culture medium. Alternatively, the polypeptides can remain in the cytoplasm or membrane fractions and the cells are harvested, lysed, and the polypeptides isolated using the desired methods.

[0243] Pharmaceutical compositions and methods of treatment

[0244] In certain embodiments, the present disclosure provides pharmaceutical compositions for administering anti-CSP antibodies and variants thereof. In certain embodiments, the pharmaceutical compositions can be administered to a mammalian subject (e.g., a human) having or at risk of malaria in a therapeutically effective amount and on a schedule sufficient to prevent infection by a Plasmodium species (e.g., an infection by P. falciparum or P. malariae having a cross-reactive CSP protein) or reduce symptoms of malaria in the subject. In certain embodiments, the pharmaceutical compositions can include any of the anti-CSP antibodies and variants thereof disclosed herein, or a polynucleotide encoding any of the anti-CSP antibodies and variants thereof, and a pharmaceutically acceptable diluent or carrier. In certain embodiments, the polynucleotide encoding the antibody can be contained in a plasmid vector (for delivery) or a viral vector. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the antibody. As used herein, “therapeutically effective dose” or “therapeutically effective amount” refers to an amount sufficient to prevent, cure, or at least partially arrest malaria or symptoms of malaria. A therapeutically effective dose can be determined by monitoring patient response to treatment. Typical benchmarks indicative of a therapeutically effective dose include improvement or prevention of malaria symptoms in the patient, including, for example and without limitation, reduction in parasite numbers. The amount effective for this use will depend on the severity of the disease and the general health of the patient, including other factors such as age, body weight, sex, route of administration, and the like. Single or multiple administrations of the antibody will be determined by the dosage and frequency as needed and tolerated by the patient.

[0245] In certain embodiments, the antibody is administered at a pre-erythrocytic stage of infection, i.e., within a time frame that prevents or reduces infection of hepatocytes.

[0246] Also disclosed herein are various pharmaceutically acceptable diluents, carriers and excipients, as well as techniques for making and using pharmaceutical compositions. Illustrative 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 must be "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition and not injurious to the subject. Generally, pharmaceutically acceptable carriers are pH buffered aqueous solutions. For example, in certain non-limiting embodiments, pharmaceutically acceptable carriers, diluents or excipients include water; buffers, 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 carboxymethyl cellulose, ethyl cellulose, cellulose acetate; kaolin; 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 laureate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethylene glycol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers 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 compositions.

[0247] In certain embodiments, the pharmaceutical composition can be formulated for any suitable route of administration, including, for example, parenteral, intrapulmonary, 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 to 100 mg / ml, 10 to 50 mg / ml, 20 to 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 to 500 mg / ml, 50 to 250 mg / ml, or 100 to 150 mg / ml, and a viscosity of less than 50 cP, less than 30 cP, less than 20 cP, or about 10 cP. In certain embodiments, the pharmaceutical composition is a liquid or a solid. In certain embodiments, the pharmaceutical composition is formulated for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration.

[0248] In certain embodiments, the formulation and method of delivery of the pharmaceutical composition are adjusted according to the site and disease to be treated. For example, without limitation, formulations include those in which the antibody is encapsulated in a micelle, liposome, or drug-release capsule (active agent incorporated into a biocompatible coating designed for slow release); ingestible formulations; formulations for topical use, such as creams, ointments, and gels; and other formulations, such as inhalants, aerosols, and sprays.

[0249] In certain non-limiting embodiments, for example, for parenteral administration, the antibody or antigen-binding fragment thereof associated with a pharmaceutically acceptable parenteral vehicle is formulated in unit-dose injections (solutions, suspensions, emulsions) in a unit-dose container. Non-limiting examples of vehicles include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as fixed oils and ethyl oleate, can also be used.

[0250] The dosage and dosage regimen are dependent on various factors easily determined by the physician, such as the nature of the infection, the characteristics of the subject, and the history of the subject. In certain embodiments, the amount of antibody or antigen-binding fragment thereof administered or provided to the 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 body weight (e.g., about 0.1 to 15 mg / kg / dose) of the antibody or antigen-binding fragment thereof can be provided to the patient as an initial candidate dosage, whether, for example, by one or more separate administrations, or by continuous infusion. Progress of the therapy is easily monitored by conventional methods and assays, and based on criteria known to the physician or other skilled artisan.

[0251] The antibodies or variants thereof of the present disclosure can be administered to a subject using any route of administration (e.g., systemic, parenteral, topical) according to known methods. Such routes include, but are not limited to, intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The subject can be administered the antibodies of the present disclosure once or multiple times; and can be administered prior to, after, or concurrently with another therapeutic agent, as further described below.

[0252] 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 infection by Plasmodium. 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 an early stage of entry into the vertebrate subject, thereby preventing the occurrence of infection.

[0253] In certain embodiments, an anti-CSP antibody of the present disclosure can be administered to treat malaria. In certain embodiments, an antibody disclosed herein can inhibit or reduce the progression of a Plasmodium infection in the bloodstream. In certain embodiments, an antibody disclosed herein can inhibit or reduce the risk of a Plasmodium being transmitted from a subject to another subject via insect feeding (e.g., mosquito bite or via contact with infected blood).

[0254] In certain embodiments, a pharmaceutical composition disclosed herein can be administered to a pediatric patient. As used herein, the term “pediatric patient” refers to a patient under the age of 18. In certain embodiments, a pediatric patient is a patient between 3 months and under 12 years of age. In certain non-limiting embodiments, a pediatric patient can be a patient between about 1 year and about 2 years, about 2 years and about 3 years, about 3 years and about 4 years, about 4 years and about 5 years, about 5 years and about 6 years, about 6 years and about 7 years, about 7 years and about 8 years, about 8 years and about 9 years, about 9 years and about 10 years, or about 11 years and about 12 years of age. In certain embodiments, a pediatric patient does not respond or responds poorly to another treatment for malaria. In certain embodiments, a pediatric patient is a human.

[0255] In certain embodiments, a dose of a pharmaceutical composition disclosed herein is administered according to the weight of a pediatric patient. In certain non-limiting embodiments, a 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, a pediatric patient has a weight of 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.

[0256] In certain embodiments, the antibody is provided to a subject in combination with one or more additional therapeutic agents for treating or preventing malaria or a related disease or disorder. In certain embodiments, a method of treating or preventing malaria is provided that includes administering a therapeutically effective amount of an antibody or a pharmaceutically acceptable salt thereof as disclosed herein in combination with a therapeutically effective amount of one or more additional therapeutic agents to a human. In certain embodiments, a method for treating malaria in a human having or at risk of having an infection is provided that includes administering a therapeutically effective amount of an antibody or a pharmaceutically acceptable salt thereof as disclosed herein in combination with a therapeutically effective amount of one or more additional therapeutic agents to a human.

[0257] In certain embodiments, when an antibody of the present disclosure as described herein is combined with one or more additional therapeutic agents as described above, the components of the composition are administered as a simultaneous or sequential regimen. When administered sequentially, the combination can be administered in two or more administrations.

[0258] In certain embodiments, an antibody as disclosed herein is combined with one or more additional therapeutic agents for simultaneous administration to a patient in a single dosage form.

[0259] A "patient" refers to any subject who receives 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 assess the effects of administration of an antibody.

[0260] "Co-administration" of an antibody disclosed herein with one or more additional therapeutic agents generally refers to the simultaneous or sequential administration of an antibody or fragment thereof disclosed herein and one or more additional therapeutic agents such that therapeutically effective amounts of both the antibody or fragment thereof disclosed herein and the one or more additional therapeutic agents are present in the patient's body. Co-administration includes administration of a unit dose of an antibody disclosed herein prior to or following administration of a unit dose of one or more additional therapeutic agents, e.g., and without limitation, administration of the antibody within seconds, minutes, or hours of administration of the one or more additional therapeutic agents. For example, in certain non-limiting embodiments, a unit dose of an antibody disclosed herein is administered first, followed within seconds or minutes by administration of 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 administration of a unit dose of an antibody. In certain embodiments, a unit dose of an antibody disclosed herein is administered first, followed after a period of time (e.g., 1 to 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 to 12 hours) by administration of a unit dose of an antibody.

[0261] Combination administration can be co-administration, using separate pharmaceutical compositions or a single pharmaceutical composition, or consecutive administration in either order, with optional overlapping of timing of administration of both (or all) therapeutic agents when their biological activities are simultaneous. Such combination treatment can result in synergistic therapeutic effects. In certain embodiments, it is desirable to administer an antibody of the present disclosure in combination with another antibody directed against another Plasmodium falciparum antigen or directed against a different CSP target epitope.

[0262] In certain embodiments, the antibody can be administered by gene therapy via a nucleic acid comprising one or more polynucleotides encoding the antibody. In certain embodiments, the polynucleotide encodes scFv. In certain embodiments, the polynucleotide comprises DNA, cDNA, or RNA. In certain embodiments, the polynucleotide is present in a vector (e.g., a viral vector).

[0263] Methods for selecting anti-CSP antibodies as therapeutic antibodies against malaria

[0264] Based on the analysis of anti-CSP antibodies as described herein, it was shown that: a) the protective activity of the antibodies in vivo was not related to the binding kinetics of the long NANP6 peptide (Table 9), but was indeed related to the k of CSP. 解离 The present invention provides a method for selecting anti-CSP antibodies (anti-malarial therapeutic antibodies) for the prevention or treatment of malaria. In certain embodiments, the method comprises analyzing the binding of an antibody to a first epitope of the central repeat region of CSP and to a second epitope of CSP that is heterologous to an epitope present in the RTS, S vaccine, wherein the antibody is selected if it binds to both the first epitope of the central repeat region of CSP and the second epitope (heterologous epitope) that is heterologous to an epitope present in the RTS, S vaccine. In certain embodiments, the method comprises: if the antibody binds to the first epitope of the central repeat region of CSP and to the second epitope (heterologous epitope) that is heterologous to an epitope present in the RTS, S vaccine, the antibody is selected. In certain embodiments, the method comprises: if the antibody binds to the first epitope of the central repeat region of CSP and to the second epitope (heterologous epitope) that is heterologous to an epitope present in the RTS, S vaccine, the antibody is selected as an anti-malarial therapeutic antibody.

[0265] 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 connecting region of CSP, including epitopes comprising DPNA.

[0266] The CSP antibodies and variants thereof disclosed herein can be selected as anti-malarial therapeutic antibodies based on their binding specificity. For example, but not limited to any limitation, the CSP antibodies and variants thereof disclosed herein can specifically bind to a first epitope (e.g., one disclosed in Table 4) and a second epitope (e.g., one disclosed in Table 5).

[0267] In certain embodiments, the method includes analyzing the binding of the antibody to a first epitope of CSP. In certain embodiments, the first epitope is contained 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 to 974. In certain embodiments, the first epitope consists of the amino acid sequence set forth in SEQ ID NOs: 923 to 974. In certain embodiments, the method also includes analyzing the binding of the antibody 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 to 1195. In certain embodiments, the second epitope consists of the amino acid sequence set forth in SEQ ID NOs: 975 to 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.

[0268] In certain embodiments, the method further comprises analyzing the binding of the antibody 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 the amino acid sequence set forth in SEQ ID NOs: 975 to 1195. In certain embodiments, the at least one additional epitope consists of the amino acid sequence set forth in SEQ ID NOs: 975 to 1195. In certain embodiments, the antibody is selected as an anti-malarial therapeutic antibody if it binds to the first epitope, the second epitope, and the at least one additional epitope.

[0269] In certain embodiments, the antibody is expressed in less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 The binding affinity (K D ) binds to the first epitope. In certain embodiments, the antibody binds to the first epitope at a rate of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 M's K Dbinds to a second epitope. In certain embodiments, the antibody binds to a second epitope with a Kd of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 M, or less than about 10 D binds to a first epitope; and binds to a second epitope with a Kd of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 M, or less than about 10 D binds to a second epitope.

[0270] In certain embodiments, the antibody further binds to a second epitope with a Kd of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 M, or less than about 10 D binds to at least one additional epitope. In certain embodiments, the antibody binds to at least one additional epitope with a Kd of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 M, or less than about 10 D binds to a first epitope; and binds to a second epitope with a Kd of less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 M, or less than about 10 D binds to a second epitope; and binds to a second epitope with a Kd of less than about 10-6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 K of M D binds to at least one additional epitope.

[0271] In certain non-limiting embodiments, K D Surface Plasmon Resonance can be used at 25°C using Immobilized antigen CM5 chips at about 10 response units (RU) were used for measurements. Briefly, after activation of the carboxymethylated dextran biosensor chip, each epitope was diluted at a consistent flow rate (e.g., 5 μl / min) prior to injection. After injection of the epitope, unreacted groups were blocked. Association rates (k 缔合 or k a ) and dissociation rates (k 解离 or k d ) were calculated using a global fitting of association and dissociation sensorgrams using a binding model. The equilibrium dissociation constant K D was calculated as the ratio k d / k a (k 解离 / k 缔合 ). Additional information on the calculation of K D can be found in Chen et al., J. Mol. Biol. 293 (1999) 865-881.

[0272] *****

[0273] From the foregoing, it will be appreciated that variations and modifications of the presently disclosed subject matter can be used to adapt the current disclosure to various uses and conditions. Such embodiments are also within the scope of the following claims.

[0274] The recitation of elements in any definition herein includes any single element or combination of elements. The recitation of embodiments herein includes embodiments as any single embodiment or in combination with any other embodiment or portion thereof.

[0275] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

[0276] All of the features of the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can 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.

[0277] The foregoing written description is considered to be sufficient to enable one skilled in the art to practice the methods and / or obtain the compositions described herein. The following examples and detailed description are provided by way of illustration only.

[0278] The disclosure of all references in the specification is expressly incorporated herein by reference.

[0279] Example

[0280] These examples are provided merely for illustrative purposes and are not intended to limit the scope of the application in any way. Indeed, various modifications of the embodiments described herein, in addition to those described herein, will be apparent to those of ordinary skill in the art from the foregoing description and accompanying drawings, and fall within the scope of the appended claims.

[0281] It should be understood that various other embodiments can be practiced in view of the general description provided above.

[0282] Example 1. Identification of functionally active anti-CSP antibodies

[0283] PBs skew towards dominant mutated immunoglobulin G lineages following RTS,S vaccination

[0284] Anti-CSP antibodies were discovered in the antibody repertoire generated by immune repertoires Technology from plasmablast B cells isolated from two donors enrolled in a Phase 2a study to evaluate the efficacy of RTS,S vaccine in preventing malaria infection. Technology and its use in antibody discovery are well known and disclosed in, for example, WO 2012148497A2, the entire contents of which are incorporated herein by reference. The RTS,S vaccine is a pseudo-virion particle vaccine that combines the hepatitis B surface antigen along with the central repeat region and C-terminal region of the CSP protein. RTS,S is composed of two polypeptides; RTS is a single polypeptide chain corresponding to amino acids 207 to 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. The RTS,S vaccine was administered with the adjuvant AS01B to improve efficacy. AS01B is a liposome-based formulation containing the immunostimulants monophosphoryl lipid A (MPL) and QS21 and was shown to be more immunogenic than another adjuvant, AS02A, used in initial studies. Kester et al., J Infect Dis 200:337-346 (2009). All study participants were vaccinated in one of two vaccine schedules (standard full dose: 0, 1, 2M or partial third dose: 0, 1, 7M) or placebo and subsequently challenged with controlled human malaria parasite infection (CHMI).

[0285] Plasmablasts (PB) were isolated from PBMC collected 7 days after the third dose (P3D; n=22, 319PB) and the fourth dose (P4D; n=10, 429PB; Table 9) prior to CHMI and used to generate naturally paired heavy and light chain IgG sequences. Almost all (99.2%) of the antibody sequences were different from the inferred germline precursor sequences. Consistent with previous malaria studies, specific germline heavy and light chain genes and pairings were frequently observed in the dataset, including IGHV3-30 / 33, KV1-5, KV3-20, and LV1-40. No significant associations were observed between the protected status and the multiple IgG sequences and repertoire features examined.

[0286] In a Phase 2a clinical trial, the immune repertoire Sequencing Platform. Messenger RNA from IgG-expressing PB isolated from peripheral blood mononuclear cells (PBMC) of individuals (n=45) vaccinated with RTS,S were sequenced on the Sequel® sequencing platform. In this trial, participants either received three full doses of RTS,S / AS01 E, one month apart (012M; n=15), or two full doses, one month apart, followed by a smaller (one-fifth, "partial") dose six months later (Fx017M, n=30). Vaccinated individuals were challenged with malaria in a controlled human malaria infection (CHMI) model after the third dose. A subset of individuals received a fourth dose of vaccine and were challenged with malaria a second time. Prior to CHMI, plasmablasts (PB) were isolated from PBMC collected 7 days after the third dose (P3D; n=22, 319 PB) and the fourth dose (P4D; n=10, 429 PB; Table 6) and used to generate naturally paired heavy and light chain IgG sequences. Almost all (99.2%) of the antibody sequences were different from the inferred germline precursor sequence Figure 4 ). Consistent with previous malaria studies, specific germline heavy and light chain genes and pairings were frequently observed in the dataset, including IGHV3-30 / 33, KV1-5, KV3-20, and LV1-40 Figures 5A to 5C ). No significant associations were observed between the protective status and examined IgG sequence and repertoire features Figures 5D to 5I

[0287] Table 6

[0288]

[0289] P3D and P4D PB were grouped into Ig lineages (n=18,980), defined here as PB sequences likely to have originated from a common progenitor B cell clone (see Example 2). Lineage sizes ranged from 1 to 84 (P3D) or 1 to 93 (P4D) PB. Since PB have a short half-life in the blood and were isolated from a small amount of blood (approximately 10 ml), detection of lineages with >2 PB indicates recent expansion of the lymphoid organ. One-fifth of lineages expanded clonally and contained at least two PB with identical or different B cell nucleotide sequences (19.4%, n=3,684 lineages, Figure 3A Consistent with antigen-driven selection pressure following vaccination, most clonally expanded lineages also showed evidence of clonal expansion Figure 3B ​), a hallmark of affinity maturation. In addition, several lineages had clonal representatives observed after the third and fourth immunizations, referred to here as “recall” lineages (4.1% to 26.6% of vaccinee P3D amplified lineages). Moreover, many of these amplified lineages also showed evidence of sequence convergence between ≥2 vaccinees when sequences were compared between vaccinees (7.3% to 46.7% of vaccinee P3D amplified lineages). Not surprisingly, lineages (n=10,841) with only a single PB observed in the P3D repertoire had significantly lower convergence rates (2.0% to 13.8%) and recall rates (1.2% to 18.6%) than amplified lineages (P<0.0001 and P<0.001, respectively, Wilcoxon matched pairs, two-tailed test) and higher levels of somatic hypermutation (SHM). Therefore, to increase the chances of identifying antibodies derived against the RTS,S antigen, subsequent analytical efforts focused on amplified lineages ( Figure 3B ).

[0290] Presumably, the lineages with the highest number of PBs per vaccinee (herein referred to as "dominant lineages") were more likely to be targeted by the vaccine because they outperformed other PB lineages in terms of antigen binding and / or T cell help in lymphoid organs. Thus, for each vaccinee, the expanded P3D lineages (9 to 99 expanded lineages were observed per vaccinee) were ranked by size ("rank size"). The sum of the PBs in the top four rank size lineages per vaccinee accounted for 17% to 100% of the total PBs in the P3D repertoire of each vaccinee's expanded lineages and 33% of the PBs in the P3D expanded lineages of all vaccinees ( Figure 3C Because this PB distribution pattern was consistent across protection states and dosing regimens, an antibody screening library was generated for in vitro and in vivo characterization that was biased toward the dominant P3D lineage in protected and non-protected vaccinees.

[0291] CSP reactivity of expanded P3D PBs is associated with lower SHM and lack of protection

[0292] One clone was selected from each of the 369 unique P3D lineages, and the gene was synthesized and recombinantly expressed for testing ( Figure 6A The library included nearly all (96%) of the largest lineages (rank size 1) in all vaccinees; approximately half (56%) of the second, third, and fourth rank-size lineages in all vaccinees; a small fraction (6.9%) of expanded subdominant lineages (rank size ≥ 5) and some single PB lineages (0.18% of 10,841 single cell lineages). All antibodies were screened in the CSP enzyme-linked immunosorbent assay (ELISA) ( Figure 3Dand 6B ), and approximately one-third (HBsAg, Figure 6C ) of the antibodies screened in both assays (n=130) reacted with CSP or HBsAg. Overall, 38% (139 / 369) bound to CSP and the binding of an additional 29 antibodies was indeterminate. Of the CSP-reactive antibodies, 73% (102 / 139) of the bound peptides were from the NANP CR region and 20 bound peptides were from the C-terminal region (data not shown).

[0293] Given that expanded lineages are more likely to show evidence of convergence and recall compared to single PB, it was tested whether these same features were associated with CSP reactivity. Indeed, antibodies from lineages that converged from >2 sequences from vaccinated individuals were more likely to bind to CSP (54%, 55 / 102) than clones from lineages that lacked evidence of convergence (31%, 84 / 267, P=0.0001, Fisher's exact, two-sided). Recall lineages were also more likely to react to CSP (49%, 43 / 87) than lineages that only observed P3D (19%, 16 / 83 P<0.0001, Fisher's exact).

[0294] As previously described, for whole sporozoite immunization, the level of SHM in CSP-reactive antibodies was found to be significantly lower than the level of SHM in CSP-negative antibodies (P<0.0001, Figure 3D ), and the level of SHM in NANP-specific antibodies was lower than the level of SHM in C-terminal binding antibodies (P<0.006, Figure 3D ). Consistent with observations on these sequence repertoires, the level of SHM in NANP-binding antibodies was independent of the vaccinated individual's protective status P3D (P>0.6, Figure 7B ). Furthermore, the percentage of antibodies specific for CSP and NANP was strikingly lower in P3D-protected vaccinated individuals than in P3D-non-protected vaccinated individuals (P<0.0007 for CSP, P<0.006 for NANP, Fisher's exact, Figure 3E and 3F ). When the analysis was restricted to antibodies from the most dominant lineages (rank size 1 to 4, P<0.0004, Figure 3G ) and when all antibodies (including the 20 antibodies from lineages that only had 1 PB) were combined in the analysis (P<0.0005, Fisher's exact and P<0.00105, by bootstrap analysis, Figure 3H) and P3D protection status. These data suggest that the quality of the CSP-specific antibody repertoire can be more important in driving protection than the total amount of circulating, CSP-specific PB or repeat-specific PB.

[0295] Subsporozoite inhibitory antibodies in P3D PB were insufficient to protect P3D.

[0296] Given this surprising inverse correlation and the extensive reporting of protective activity of CSP-binding antibodies in humans and mice, antibodies were selected as potential anti-malaria prophylactics to advance without assuming any protection correlation. Seventy-seven antibodies (77 unique lineages) were selected, including NANP-reactive antibodies and C-terminal reactive antibodies from both protected (n=26) and unprotected (n=8) vaccinees, as well as dominant and subdominant lineages from high or low SHM levels (>20 or <20 nucleotide mutations from the germline, respectively, for each antibody). Since in vitro functional assays have demonstrated limited predictive power for in vivo anti-malaria activity, the mouse sporozoite infection model was used to screen the antibodies for activity. More than half of these antibodies (44 / 77) provided >95% inhibition of sporozoite liver burden, and some provided near complete protection (>99.9% inhibition). All 44 antibodies bound to the NANP repeat region of CSP, most were derived from the IGHV3-33 germline, some from other IGHV3 genes, and one from IGHV1. Thirteen additional NANP-binding IGHV3-30 / 33 antibodies showed limited inhibition (80% to 95%) of parasite liver burden, and 12 antibodies, including three C-terminal peptide binders, showed minimal but detectable inhibition (20% to 80%, <95% inhibition), and one antibody showed no inhibition of sporozoite liver burden. Figure 1A ) in mice.

[0297] Approximately one-third of the test antibodies (30%, 23 / 77) were from unprotected vaccinees, half of which (7 / 14) showed near complete protection (>99.9% inhibition, Figure 1A ) in mice. These data suggest that expression of these inhibitory antibodies by circulating, amplified P3D PB lineages was insufficient to drive protection. For example, the highly potent antibody AB-000317 Figure 3HHowever, the PB expansion level of this antibody lineage differed between the two vaccinees. In the protected vaccinee, this antibody was a member of the largest PB lineage, while in the unprotected vaccinee, it was expressed in the seventh largest size lineage (15.8% and 1.7% of PB, and 10% and 0.9% of all circulating P3D PB, in the expanded P3D lineage, respectively). These data are consistent with the hypothesis that, in addition to the functional activity of the antibodies, the number of PB expressing the antibodies can influence the state of protection, ultimately by affecting the titer in the blood and / or the performance in the immunological memory.

[0298] Inhibitory antibodies from vaccinees do not bind to CSP peptides not present in RTS,S

[0299] To explore the developability of these inhibitory antibodies as potential drugs, 35 NANP repeat binding lineages were selected from the 52 that demonstrated >90% inhibition in the sporozoite challenge screen for further pharmacological studies. To avoid sequence signatures that can become burdensome in the antibody drug development process, and to investigate the clonality of lineages with broad clonal diversity, more than one unique antibody clone was selected from some (n=23) lineages. Overall, up to 141 antibodies (representing a range of high and low SHM levels) from 21 protected vaccinees of both RTS,S dosage regimens were tested in binding assays.

[0300] Antibodies exhibit a broad range of affinities for CSP (K D ranging from 11 pM to 9.8 nM, Figure 1B , Tables 7 and 8). While there was a negative correlation between the vaccinee protection status and the percentage of CSP-reactive antibodies observed in the original screening library Figures 3E to 3H , these selected inhibitory antibodies had a significant association between CSP binding affinity (K D ) 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 post-vaccination. These correlations can be driven by two relationships: the association rate (k 缔合 ) with CSP and heavy and light chain SHM levels (Figures 10A and 10B, Table 9) and the dissociation rate (k 解离 ) with CSP and SHM levels in the light chain (P<0.0005, r=-0.29, Spearman; Table 9).

[0301] Table 7

[0302] Table 7 | Binding and in vivo pharmacology of lead mAbs

[0303]

[0304]

[0305]

[0306]

[0307]

[0308] Table 8

[0309] Table 8 | Binding and in vivo pharmacology of lead mAbs

[0310]

[0311]

[0312] Table 9

[0313]

[0314] nd, not done; NA, not applicable; LB, liver burden mouse model; NPNA3, NPNANPNANP; linked, KQPADGNPDPNANPN; NPDPNANP2NVDP, NPDPNANPNVDPNANP; NVDP3NANP2, NVDPNANPNVDPNANPNVDP; NANP6, NANPNANPNANPNANPNANPNANP

[0315] a SPR assay K 缔合 (K a ), K 解离 (K d ), and K D , see Methods

[0316] b S, Spearman correlation; P, Pearson correlation; LR, K 解离 K 缔合 or KD data

[0317] c Liver burden inhibition normalized to percent inhibition of AB-000317 tested in parallel

[0318] The binding of antibodies to short (12 to 15 residues) and long peptides (20 to 24 residues) derived from various tetrapeptide-based homologous (NPNA3 and NANP6 peptides) and heterologous epitopes (NPDPNANPNVDPNANP, NVDP3NANP2, and the linking [KQPADJNPDPNANPN] peptide) of CSP was also evaluated ( Figure 1B The strongest correlations observed were for SHM with short major repeat peptides, short minor repeat peptides, and JR peptides. 解离 The negative correlation between Figures 1C to 1E ). K calculated for long homologous and heterologous peptides and for CSP 解离 The correlation between the incidence rate and SHM level was weak, but still statistically significant, or not significant at all ( Figures 1F to 1G , Table 9). Indeed, the strongest correlation was observed between SHM and binding rates for short cognate peptides, even though the long versions of the cognate peptides contained more repeats of the same epitope (Table 9).

[0319] Furthermore, the correlation between SHM levels and the binding rate of JR peptides (heterologous to the epitope in RTS,S) was stronger than that of long homologous peptides (Table 9). These data suggest that B cell receptor maturation of these highly functional antibodies may be driven preferentially by interactions with short versus long NANP epitopes, which favor maturation of heterologous peptide sequences. These observations are consistent with reports that protective antibodies from anti-CSP immune responses can exhibit promiscuous binding between different CSP epitopes, although other reports suggest that such promiscuity is not necessarily required for protection.

[0320] Anti-sporozoite activity correlates with CSP peptide binding and SHM levels

[0321] Seventy antibodies (representing 33 of the 35 protective lineages evaluated in the binding study) were directly compared with the highly potent antibody AB-000317 in an intravenous sporozoite challenge mouse model. The antibodies inhibited sporozoite liver burden by 44.1% to 97.5% (47.4% to 103.8% inhibition by AB-000317, Tables 7 and 8). Overall, approximately half of the antibodies exhibited comparable inhibition to AB-000317 (n=32), while the other half exhibited significantly weaker inhibition (n=36), and AB-000224 exhibited superior activity to AB-000317 (n=36). Figures 2A to 2B , Tables 7 and 8). The serum concentrations of most antibodies were at least higher than the CSP K values ​​of the corresponding antibodies. D 1000 times higher ( Figure 2C , Tables 7 and 8), suggesting that antibodies are unlikely to exhibit weak inhibition due to low levels of circulating antibodies. Lineages with at least one antibody that exhibits activity consistent with AB-000317 are considered for further advancement.

[0322] To determine whether RTS, S-driven affinity maturation contributed to antibody inhibition, we assessed whether percent inhibition compared to AB-000317 correlated with peptide binding kinetics or SHM levels. Relative activity correlated with slower k 解离 ( Figure 2D ) from CSP, slower k 解离 ( Figure 2E ) from the short homologous peptide NPNA3, and slower k 解离 ( Figures 2F to 2G , Table 9) from JR and another short heterologous peptide. Notably, no significant correlation was observed between inhibitory activity and binding kinetics for the long homologous peptide NANP6 (P > 0.3 [k 解离 ]; P > 0.7 [k 缔合 ], Spearman and Pearson, Table 9), despite this peptide being the most representative in both RTS, S and CSP. Taken together, the data evaluating these inhibitory antibodies suggest that while binding to the NPNA epitope can be required, mutations that favor binding to heterologous peptides can be superior to mutations that only improve binding to the longer homologous NPNA epitope.

[0323] Affinity maturation via SHM can underlie the correlation between in vivo function and binding kinetics, as inhibitory activity significantly correlated with changes in heavy and light chain nucleotides and amino acids from the germline ( Figures 2H to 2I , Table 9). Consistent with this observation, low SHM antibodies were more likely than antibodies with higher mutational loads to exhibit significantly weaker inhibition compared to AB-000317 (86% [12 / 14] vs. 44% [24 / 55], P = 0.0069; Fisher’s Exact, two-sided). Taken together, these correlations between higher SHM levels and binding kinetics for homologous epitopes ( Figure 1C and 1F ) and heterologous epitopes ( Figures 1D to 1E and 1G), and between higher SHM levels and inhibitory activity ( Figures 2H to 2I ), suggest that affinity maturation to the RTS, S epitope includes bystander maturation to heterologous epitopes, which can be functionally important.

[0324] Despite the correlations between SHM levels, inhibitory activity, and k 解离 for CSP and short peptides, some antibodies with high SHM levels were exceptions. In some cases, high SHM antibodies had relatively fast k 解离 and slower k 缔合 , and were relatively poor inhibitors like many low SHM antibodies ( Figure 2J). These antibodies can be due to inefficient affinity maturation, affinity maturation leading to lower inhibitory paratopes, and / or abnormal selection mechanisms that limit survival and recall from memory Figures 3E to 3H ). In other cases, some high SHM antibodies have relatively faster k 解离 and slower k 缔合 , despite their unfavorable binding kinetics, are still relatively good inhibitors Figure 2J . In the latter case, affinity maturation to inter-Fab-Fab interactions (rather than CSP epitopes) can contribute to relatively strong activity. Inter-antibody binding events can contribute to anti-CSP binding potency and increase functional activity, and some of the antibodies described here have been reported. Such inter-Fab interactions can not be reflected in binding kinetics to short NPNA3 peptides, which are too short to sterically accommodate multiple simultaneous binding events. Indeed, four antibodies with relatively faster k 解离 , but comparable activity to AB-000317, are from lineages containing antibodies that bind via inter-Fab-Fab interactions (AB-000399, Figure 2J , red circles). Overall, this data is consistent with affinity maturation of antibodies via multiple different binding modes, and reveals several antibodies (>30) with comparable activity to AB-000317 that can be developed as clinical leads.

[0325] Using sporozoite liver burden data, this example further down-selected 26 mAbs representing 15 lineages for evaluation in the parasitemia challenge model as a surrogate endpoint to assess function in vivo. This set included AB-000317, AB-000224, 23 additional mAbs with liver burden inhibitory activity similar to AB-000317, and one mAb with weaker activity than AB-000317. Except for two mAbs, all mAbs were more likely than the negative control to prevent parasitemia. Compared to AB-000317, seven mAbs (including AB-000224 and two additional mAbs from the same lineage) showed a better trend toward protection (non-parametric log-rank hazard ratio < 1 vs. AB-000317, Figure 2K ; Tables 10-12). At infection, serum concentrations of nearly all mAbs (25 / 26) were at least 1000-fold higher than the KDCSP-SPR of the corresponding mAb (Tables 7 and 8), suggesting that mAbs more potent than AB-000317 can not be missed due to low circulating antibody levels.

[0326] Table 10

[0327]

[0328] Table 11

[0329]

[0330] Table 12

[0331] Example 2. Method

[0332] This example provides details of the methods and experimental strategy used to produce the results shown in Example 1 above.

[0333] Vaccine recipients, plasmablast isolation, IgG sequencing

[0334] PB's collection is RTS, S / AS01 (Mosquirix TM The study was conducted as part of a Phase 2a clinical trial of the third and fourth doses of the 2019-nCoV vaccine. The protocol 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 participant before initiation of study procedures (ClinicalTrials.gov identifier: NCT01857869). Unique samples of PBMCs obtained from trial participants for this study were used exhaustively and are not available for analysis.

[0335] B cell isolation, cloning, and sequencing were performed using the following publicly available protocol. PBMC were stained with the following mAbs: anti-CD3-FITC (BioLegend, Cat# 300406, clone UCHT1), anti-CD14-FITC (BioLegend, Cat# 325604, clone HCD14), anti-CD19-BV421 (BioLegend, Cat# 302234, clone HIB19), anti-CD20-PerCP / cy5.5 (BD, Cat# 340955, clone L27), anti-CD27-BV510 (BioLegend, Cat# 302836, clone O323), anti-CD38-PE / cy7 (BioLegend, Cat# 356607, clone HB-7), anti-IgA-FITC (Miltenyi, Cat# 130-113-175, clone IS11-8E10), anti-IgM-APC / cy7 (BioLegend, Cat# 314520, clone MHM-88). IgG+ PB single cells were sorted into 96-well PCR plates containing hypotonic buffer (330 nM dNTPs (NEB, Cat# N0447L), 1 pg / ml BSA (NEB, Cat# B9000S), 2 mM DTT (Sigma-Aldrich, Cat# 43816), 0.5% IGEPAL-430 (Sigma-Aldrich, Cat# I8896), and 200 units / ml Ribolock (Thermo Fisher Scientific, Cat# EO0384)) based on gating for CD3-CD14-CD19+CD20-CD27+CD38++IgA-IgM- cells. IgG mRNA isolated from single cell sorted PB was sequenced with the following modifications: reverse transcription was performed using desulfurated biotinylated oligos (dT) and Maxima H-Reverse Transcriptase (Thermo Fisher Scientific, Cat# EP0753), cDNA was extracted using MyOne TM MyOne TM C1 streptavidin beads (Thermo Fisher Scientific, Cat# 65001), qPCR (KAPA Biosystems) was used to determine the concentration of final NGS library preparations, and native paired IgG heavy and light chain amplicons were sequenced using Roche FLX+ 154 Titanium. rapid qPCR kit, Kapa Biosystems) and native paired IgG heavy and light chain amplicons were sequenced using Roche FLX+ 154 Titanium.

[0336] DNA barcode assignment and sequence assembly were performed as described: a minimum coverage of 10 reads per heavy and light chain assembly was required for acceptance. Intra-well assembly of unique contigs required both heavy and light chain reads. In the presence of more than one contig, unless one of the contigs included at least 90% of the reads, the well was rejected for consideration.

[0337] Sequence, lineage, and repertoire feature analysis

[0338] Determination of germline assignment and SHM levels

[0339] Assignment of variable (V), diversity (D), and joining (J) gene segments and mutation identification were performed using somatic diversification analysis (SoDA)65and implementation of IMGT_20203166 of the IMGT human immunoglobulin germline database. SHM substitutions were calculated for each antibody by aligning the heavy and light variable domains (start of framework 1 to end of framework 4) to a hidden Markov (Markov) model that included states for the germline aligned region and N nucleotide region (VDJ for heavy, VJ for light) and only substitutions relative to the germline sequence were calculated at the aligned portion (excluding rare, observed indels). IgG isotype (IgG1-4) assignment was performed by aligning sequence 3’ of framework 4 to IMGT human Ig constant region sequences from IMGT_20203166.

[0340] CDR3 and lineage assignment

[0341] Complementarity determining region 3 (CDR3) sequences were defined by the Kabat annotation plus the first amino acid residue of framework 4 from which CDR3 length was calculated. Clones of naturally paired IgG sequences were assigned to the same lineage if they originated from the same vaccinated individual, had identical IGHV and IGK / LV germline gene assignment, identical heavy chain CDR3 (H3) length, identical light chain CDR3 (L3) length, and at least 75% nucleic acid sequence identity between the linked H3 and L3. In certain cases, clones with IGHV3-33 and IGHV3-30 (germline genes with high sequence identity) met all criteria for assignment to the same lineage except for the difference in IGHV. In these cases, the clones were assigned to the same lineage. Lineages were assigned a rank size according to the lineage frequency (number of PB expressing the clone in the lineage divided by the total number of PB in the repertoire). In certain cases, more than one lineage in the repertoire had the same rank size because the lineages had the same number of PB.

[0342] Convergence, clonality and recall

[0343] Two IgG clones are defined as convergent if they originate from different vaccinees, have identical IGHV and IGK / LV germline gene assignment, identical H3 length, identical light chain CDR3 (L3) length, and at least 85% BLOSUM62 weighted amino acid sequence identity between the linked H3 and L3. A lineage is defined as convergent to another lineage if it originates from different vaccinees and at least one IgG clone in the first lineage converges with at least one IgG clone in the second lineage. Clonality is summarized as the normalized entropy of all lineages in each P3D vaccinee repertoire. Specifically, the sum over i of -(Ki / N*log(Ki / N)) / log(N), where N equals the number of lineages in the repertoire and Ki is the size of each lineage as number of PBs, with i being 1 to N. The value of normalized entropy is between 0 and 1, where 0 indicates that a single lineage is completely dominant in abundance and 1 indicates that some set of lineages N>1 are equally abundant. Recalled lineages are defined as lineages in which at least one PB antibody clone was observed in the P3D and P4D repertoires of a vaccinee (n=17) from which at least 100 PBs were sequenced from the P4D PBMC sample.

[0344] Lineage and clone selection of the CSP ELISA screened library

[0345] PB lineages (n=369) selected for CSP reactivity initial screening as described in Example 2, and specific clones from each selected lineage were selected for recombinant expression and screening according to the following >1 properties: i) the clone has paired heavy and light chain amino acid sequences that are expressed by more PBs in the lineage than any other clone (“dominant clone”); and / or ii) the selected specific antibody sequence converges with at least one other specific antibody sequence in a lineage from another vaccinee, where convergence is defined by the methods described in “Convergence, Clonality, and Recall” above (“convergent clone”); and / or iii) the clone is a “multi-leaf descendant” identified from the phylogenetic tree of the lineage, where the terminal branches of the leaves are ordered according to their leaf number, with the largest terminal branch defined as multi-leaf (“multi-leaf descendant clone”); and / or iv) the clone has the most number of germline nucleic acid mutations among all clones in the lineage (“mutation most clone”). Three clones (0.8% of the screened library) did not meet any of these criteria because errors were detected only after screening had occurred. The proportion of antibodies meeting each criterion from protected and unprotected vaccinees was not statistically different from the proportion of all antibodies in the screened library, Fisher’s exact.

[0346] Recombinant antibody production

[0347] 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 heavy chain IgGl and appropriate human kappa or lambda light chain constant region encoding domain sequences. Each completed construct was sequence confirmed prior to proceeding with DNA plasmid production scale-up. Suspended HEK293 cells were seeded in shake flasks and expanded using serum-free chemically defined media. On the day of transfection, expanded cells were seeded into new flasks with fresh media. Each DNA construct was transiently transfected into HEK293 cells using a cationic lipid transfection method. Cells were maintained as a batch fed culture until the end of the production run. Conditioned media from transient production runs was harvested and clarified by centrifugation and filtration. 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 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 pm membrane filter. Purified antibody was dialyzed against PBS and analyzed using LabChip GXII. Endotoxin measurement was performed using a chromogenic Limulus Amebocyte Lysate method with Pyrochrome (Associates of Cape Cod)

[0348] CSP, NANP peptide, and C-terminal peptide ELISA

[0349] Antibodies were mapped to CSP using near full-length CSP, (NANPx6) peptide, and CSP C-terminal peptide (Pf16). For the purposes of this study, antibodies were classified as "positive," "negative," or "equivocal." All antibodies were evaluated at a concentration of 0.15 or 0.04 pg / ml. ELISA optical density (OD) was converted to fold induction of the mean value of four negative control antibodies run in each experiment. The "equivocal" range of the experiment was then determined using the range of OD responses observed in each experiment. Antibody OD that failed to exceed the mean negative control antibody OD + 3x standard deviation was classified as "negative." Antibodies with OD higher than the mean negative control antibody OD + 3x standard deviation but that had not yet exceeded the negative threshold + 20% of the experimental OD value range were classified as "equivocal." Any antibody OD above the experimental "equivocal" threshold was classified as "positive."

[0350] HBsAg ELISA

[0351] The MONOLISA Anti-HB EIA kit [Bio-Rad Cat# 25220] was used to determine antibody reactivity to HBsAg. A four-point 1:3 dilution series was prepared for each test article in duplicate. The maximum input of each of the 139 test antibodies was 10% of the total volume of purification. If the total volume of purification <10% was required, the starting concentration of each test antibody was adjusted individually to 300 nM. Otherwise, the starting concentration was based on the amount of protein included in 10% of the total volume of purification. The cutoff calibrator from the kit was performed in quadruplicate, while two negative controls were each performed in duplicate. A four-point 1:3 dilution dose-response curve of PC3 starting at 150 pM was also performed in duplicate. Antibodies were considered HBsAg positive if the signal met the cutoff calibrator criteria at at least one concentration <30 nM. Antibodies were considered “borderline” HBsAg reactive if the signal was negative at test concentrations <30 nM, but met the cutoff calibrator criteria at any concentration >30 nM. Antibodies were considered negative if the signal did not meet the cutoff calibrator criteria at any test concentration.

[0352] Selection of mAbs for initial characterization in a mouse sporozoite challenge model

[0353] Of the 102 antibodies that reacted in the NANP6 peptide ELISA, Figure 1D Of the 102 antibodies that reacted in the NANP6 peptide ELISA, 69 were initially selected for in vivo screening based on representation of IGHV, vaccine protection status, and level of SHM. Specifically, the 102 antibodies were divided into 11 groups based on the 11 different IGHVs expressed therein (IGHV1-2, 1-69, 1-8, 3-15, 3-23, 3-30, 3-33, 3-48, 3-49, 3-7, and 5-51), and at least half of the mAbs in each group were selected, including mAbs from both protected (n=26) and unprotected (n=8) vaccines. Except for the antibodies containing IGHV3-33 and 3-49, only antibodies with high levels of SHM (≥20 nucleotide mutations from germline per antibody) were selected, with some of the antibodies with low levels of SHM (<20 nucleotide mutations from germline per antibody) included as well. Two of these selections did not express enough material to be tested in vivo (the only antibody containing IGHV3-23 and one of the two antibodies containing IGHV5-51).

[0354] Of the 20 antibodies reactive in the C-terminal (Pf16) peptide ELISA, 11 of the 12 antibodies from protected vaccinees were initially selected for in vivo screening. These included all IGHV germline genes observed in the C-terminal (Pf16) binders of protected vaccinees (IGHV3-11, IGHV3-21, IGHV3-30, IGHV3-48 and IGHV4-59). One of these selections did not express sufficient material to proceed to in vivo testing (the only mAb containing IGHV3-11).

[0355] Selection of antibodies from the library for use in surface plasmon resonance (SPR) binding analysis

[0356] Of the 52 mAbs representing 52 unique lineages, all mAbs derived from protected vaccinees (n=36) were selected for further binding analysis in the initial sub- sporozoite liver burden mouse model screen showing >90% inhibition, except for one mAb (AB-000239) reactive in the HBsAg ELISA. For each of the 35 antibodies representing 35 unique lineages, the original hit antibody was selected if it did not contain high-risk liabilities (i.e. odd number of cysteines in CDRs, any canonical N-linked glycosylation site in CDRs, Fv net charge (pH 5.5) > 9 or Hydrophilicity Index > 6.5). In the case of original hit liabilities > 1, additional clones were selected from the lineage of the P3D or P4D PB repertoire. Furthermore, more than one clone was selected from lineages with broad clonal sequence diversity. This was done using the following algorithm: i) query each clone of a lineage in order of multilocus offspring; ii) skip any clone with high-risk liabilities > 0; iii) skip any clone that is too close to any already selected clone, where the distance between clones was determined as the fraction of CDR amino acids that are not conserved between clones using the BLOSUM62 matrix (< 0); iv) adjust the acceptable distance between clones so that a total of 141 clones were ultimately selected from the 35 lineages.

[0357] High-throughput SPR

[0358] Binding kinetics measurements of antibody interactions with CSP antigens were performed at 25 °C using a Carterra LSA high-throughput SPR platform and CMD200M sensor chips (Carterra). The antigen panel included recombinant CSP and synthetic peptides NPNA3 (NPNANPNANPNA), NANP6 (NANPNANPNANPNANPNANPNANP), Linker peptide (KQPADGNPDPNANPN), NPDPNANP2NVDP (NPDPNANPNVDPNANP), and NVDP3NANP2 (NVDPNANPNVDPNANPNVDP) custom made by CPC scientific. All other peptides were acetylated at the N-terminus and amidated at the C-terminus except for NANP6 which contains an N-terminal biotin-aminohexanoic acid tag and an unmodified C-terminus. Two microfluidic modules, a 96-channel print head (96PH) and a single flow cell (SFC) were used to deliver liquids onto the sensor chip. In each assay, a single analyte antigen was titrated against immobilized antibody.

[0359] Antibody immobilization on the CMD200M chip depends on the type of analyte used during titration. In assays involving 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) mixed 1 : 1 : 1 with 0.1 M MES buffer at pH 5.5 for 400 seconds, then the anti-human IgG Fc was immobilized at 50 pg / ml in 10 mM sodium acetate at pH 4.5 for 900 seconds. Non-reactive esters were quenched with a 1 M ethanolamine-HCl injection at pH 8.5 for 400 seconds. The chip was then exposed to a double pulse of 10 mM glycine at pH 2.0 (30 seconds per pulse). Then, using the 96PH, antibodies were injected at a concentration of 10 pg / ml or 5 pg / ml for 400 seconds using IX 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, capturing CSP-specific antibodies on the anti-Hu IgG Fc surface. If CSP peptide antigens were used as the analyte, the chip was activated by NHS / EDC for 400 seconds, then CSP-specific antibodies were injected directly at a concentration of 10 pg / ml or 5 pg / ml for 400 seconds using the 96PH in 10 mM sodium acetate at pH 4.5. Non-reactive esters were then quenched with a 1 M ethanolamine-HCl injection at pH 8.5 for 400 seconds. Then, 45 cycles of IX HBSTE buffer injection were used, where IX HBSTE also served as the running buffer, washing off overnight non-specifically bound IgG from the sensor chip surface without the use of a regeneration buffer. The running buffer, in addition to capturing antibodies via anti-human IgG Fc and washing non-specifically bound IgG, was 10 mM MES buffer at pH 5.5 with 0.01% Tween-20. Steps were completed using the SFC unless otherwise specified.

[0360] During initial screening, each antibody at a given dilution concentration was immobilized on two independent spots on the same chip, enabling repeated measurements of binding kinetics. For binding measurements of engineered variants, each antibody was immobilized on three different spots, enabling triplicate measurements.

[0361] A two-fold dilution series of antigens was prepared in lx HBSTE buffer. The highest concentration of full-length CSP and all CSP peptide antigens was 8 pg / ml (0.25 pM for CSP, 2.92 pM for NANP6, 6.41 pM for NPNA3, 3.76 pM for NVDP3NANP2, 4.70 pM for NPDPNANPNVDPNANP, and 5.03 pM for N-Interface). Then, different concentrations of antigens were injected from the lowest to the highest concentration on the chip surface using SFC without regeneration, including eight injections of buffer before the lowest non-zero concentration to stabilize the signal. For each concentration, data collection involved a 120-sec baseline step and a 900-sec dissociation step. The association step duration was 240 sec for full-length CSP and NANP6 antigens and 300 sec for all other CSP peptide antigens. For all assays, the running buffer for the titration was lx HBSTE.

[0362] The collected kinetic titration data were first pre-processed in NextGenKIT (Carterra) software, including reference subtraction, buffer subtraction, and data smoothing. The data were then exported and analyzed using an in-house developed titration analysis tool 68. The specific binding time course of each antibody construct immobilized at different points was fitted to a 1 : 1 Langmuir model to get ka (“kon”), kd (“koff”), and KD values. The KD values for antigen assays with multiple epitope repeats include avidity effects. The average of replicate measurements for each antibody-antigen pair from the initial screening panel was reported. For engineered variants, the average of triplicate measurements was reported with the following data acceptance criteria: i) the standard error of estimated kon, koff, and KD in each replicate < 20%, and ii) the fold change of all three parameters < 3 across triplicates.

[0363] In vivo functional assessment

[0364] The assays using mice were performed strictly following the recommendations in the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. The protocol was approved by the Johns Hopkins University Animal Care and Use Committee, protocol numbers MO18H419 and MO21H417

[0365] Subsporozoite challenge liver burden mouse model Figure 1D

[0366] Initial functional screening was performed on mice immunized with anti-CSP antibodies and challenged intravenously 16 hours later with 2,000 P. berghei transgenic sporozoites expressing the complete P. falciparum CSP. Forty-two hours later, mice were euthanized and their livers excised for RNA extraction, RTqPCR was performed using forward primer 5'-TGGGAGATTGGTTTTGACGTTTATGT-3' and reverse primer 5'-AAGCATTAAATAAAGCGAATACATCCTTAC-3' to measure Plasmodium 18s rRNA. Parasite burden was expressed as P. berghei 18s rRNA copy number and the percentage of inhibition of the burden compared to the negative control was calculated.

[0367] All other liver burden assays were performed as described by Flores-Garcia et al.20Briefly, transgenic P. berghei sporozoites expressing P. falciparum CSP and luciferase infected Anopheles mosquitoes were kept in an incubator at 19°C. Mosquitoes were harvested for sporozoites in HBSS-FBS 2% at day 20 to 23 post-infection. Mice were administered 100 pg of antibody per mouse (passive immunization) and challenged 16 hours later with 2000 sporozoites injected intravenously. Control mice received irrelevant antibodies or no antibodies. Forty-two hours post-challenge, mice were injected with 100 mΐ D-luciferin (30 mg / ml), anesthetized with isoflurane and bioluminescence expressed by the parasites in the liver was measured using an IVIS Spectrum Imager Perkin Elmer. Results were expressed as photons / second.

[0368] Mosquito bite challenge of a parasitemia mouse model

[0369] Mosquito bite challenge to assess sterile protection was performed as described by Flores-Garcia et al. Briefly, 7 to 8 weeks old mice were passively immunized with 150 pg / mouse of the indicated antibodies, 16 hours later, mice were anesthetized and placed on top of cages containing five mosquitoes infected with P. berghei sporozoites expressing P. falciparum CSP and luciferase for 10 minutes. From day 4 to day 10 post-challenge, blood smears stained with Giemsa were observed under an optical microscope to determine the appearance of parasitemia. Control mice receiving irrelevant antibodies or no antibodies were also subjected to similar challenges.

[0370] Assessment of mAb concentration in serum samples

[0371] Capture antibody (AffiniPure Mouse Anti-Human IgG Fc fragment specific, Jackson ImmunoResearch #209-005-098) was adsorbed to a 96-well polystyrene microplate (Immuno Plate Maxisorp, ThermoFisher Scientific #439454) in PBS [Dulbecco's Phosphate-Buffered Saline, without calcium and magnesium, sterile pH 7.4, Wisent #311-425-LL] overnight at 21 °C and then washed 3 times in wash buffer (0.05% TWEEN 20 [Sigma #P2287] in PBS). 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 3 times with wash buffer, mouse serum samples and control standards were added in duplicate in normal mouse serum in serial dilution, then further diluted 100-fold in assay buffer before incubation for 1 hour at 21 °C. Control standards consisted of AB-000317 serially diluted from 0.146 to 25.6 ug / ml in 1.6-fold increments. The microplate was then washed 3 times with wash buffer and incubated with mouse monoclonal anti-human IgG antibody conjugated to horseradish peroxidase (HRP-conjugated clone JDC-101, Southern Biotech #9040-05) in assay buffer for 1 hour at 21 °C. After washing 3 times with wash buffer, peroxidase substrate TMB (Bio-Rad #1721068) was added, followed by stop solution (TMB Stop Solution [650 nm], Southern Biotech #0413-01L). Absorbance was measured at 650 nm (using a Molecular Devices microplate reader with SoftMax Pro GxP version 6.5.1), and the concentration of human IgG in the test samples was calculated by interpolating the OD values on the 5-parameter logistic standard curve (average OD derived from duplicate standard samples) using the standard curve generated from the control antibody, and adjusted according to their corresponding dilution factors. The final sample concentration was then determined by calculating the average of all concentrations obtained for the sample in the range of the standard curve.

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Claims

1. A recombinant anti-circumsporozoite (CSP) antibody that 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 according to claim 1, wherein the first epitope comprises the amino acid sequence NPNA.

3. The recombinant antibody according to claim 1 or 2, wherein the first epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 923 to 974.

4. The recombinant antibody according to any one of claims 1 to 3, wherein the second epitope is heterologous to an epitope present in the RTS,S vaccine. The recombinant antibody according to any one of claims 1 to 4, wherein the second epitope comprises the minor repeat region of CSP and / or the connecting region of CSP. The recombinant antibody according to claim 5 , wherein the second epitope comprises a minor repeating amino acid sequence containing DPNA / NPNV and / or a connecting amino acid sequence containing DPNA / NPNV.

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 NO: 975 to 1195.

8. The recombinant antibody according to any one of claims 1 to 7, wherein the antibody binds to at least one additional epitope of CSP. 9 . The recombinant antibody according to claim 8 , wherein the at least one additional epitope comprises a minor repeating amino acid sequence containing DPNA / NPNV and / or a connecting amino acid sequence containing DPNA / NPNV. 10 . The recombinant antibody according to claim 8 , 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 according to 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 according to claim 1 , comprising a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 461.

13. The recombinant antibody according to 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. The recombinant antibody according to any one of claims 1 to 7, comprising a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 462 to 922.

15. The recombinant antibody according to any one of claims 1 to 7, comprising: VH comprising an amino acid sequence at least about 80% identical to an amino acid sequence selected from SEQ ID NOs: 1 to 461; and 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. The recombinant antibody according to any one of claims 1 to 7, comprising: VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 461; and VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 462 to 922.

17. The recombinant antibody according to any one of claims 1 to 7, comprising 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 sequences 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 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) and a light chain variable region (VH) listed in Table 3.

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 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. 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 according to any one of claims 1 to 23. An expression vector comprising the polynucleotide according to claim 24 .

26. A host cell comprising the polynucleotide according to claim 24 or the expression vector according to claim 22.

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

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

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

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

32. The recombinant antibody according to any one of claims 1 to 23 or the composition according to claim 27 or 28 for use in preventing or treating malaria in a subject in need thereof.

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

34. Use of the recombinant antibody according to any one of claims 1 to 23 or the 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. The use according to claim 34, wherein the patient is a pediatric patient.

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

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

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

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

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

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

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

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

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 the group consisting of 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 method of any one of claims 36 to 46, wherein the antibody is expressed in an amount less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 The binding affinity (K D ) binds to the first epitope.

48. The method of any one of claims 36 to 47, wherein the antibody is expressed in an amount less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 M's K D Binds to the second epitope.

49. The method of any one of claims 36 to 48, wherein the antibody a) with less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 M's K D binds to the first epitope, and b) with a concentration less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 M's K D Binds to the second epitope.

50. The method of claim 37 or 39, wherein a) the first epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 923 to 974; b) the second epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 975 to 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 to 1195.

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

52. The method of any one of claims 36 to 48, wherein the antibody a) with less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 M's K D binding to the first epitope; b) with a concentration less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 M's K D binds to the second epitope; and c) with a concentration less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 M's K D Binds to the at least one additional epitope.

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