Antigen binding proteins and uses thereof

Abstract

The present invention provides antigen binding proteins, such as antibodies that target Plasmodium sporozoites, in particular Plasmodium circumsporozoite proteins. The invention also provides nucleic acids encoding such antibodies. In addition, the invention provides an antibody according to the invention for use in the prevention of malaria.

Description

Technical Field

[0001] The present invention relates to the field of malaria drug treatment, in particular to antibodies that bind to Plasmodium falciparum sporozoites, in particular to circumsporozoite proteins of Plasmodium falciparum, for preventing malaria. Background Art

[0002] Malaria is one of the most serious public health problems in the world. Malaria is caused by parasitic protozoa of the genus Plasmodium. The genus Plasmodium includes about 200 species, of which P. falciparum, P. vivax, P. ovale and P. malariae together cause almost all human infections of Plasmodium species. Among these Plasmodium species, P. falciparum causes the vast majority of malaria deaths, especially in children under five years old. Malaria symptoms usually include fever, feeling tired, vomiting and headache. In severe cases, it can cause yellowing of the skin, seizures, coma or death.

[0003] Malaria is a mosquito-borne disease transmitted by the bite of infected female Anopheles mosquitoes. In the case of human malaria caused by Plasmodium falciparum infection, female Anopheles mosquitoes inject a small amount of sporozoites (10-100) into the skin. Some of these parasites go to the liver to invade hepatocytes (Crompton et al. (2014) Annu Rev lmmunol 32, 157-187). In hepatocytes, these sporozoites form parasitic vacuoles, where they develop, reproduce asexually (organize schizogony) and mature into schizonts, which rupture when mature to release thousands of merozoites, which are eventually released into the bloodstream. Merozoites infect red blood cells, mature from rings to trophozoites and eventually form schizonts, which rupture to release merozoites, which will infect new red blood cells with a cycle of 48 hours, allowing the cycle to continue. Other merozoites develop into sexual erythrocytes (gametocytes). When a mosquito bites an infected person, the gametocytes are ingested with the blood and mature in the mosquito gut. The male and female gametocytes fuse and form a kinetochore, a fertilized, motile zygote. The kinetochore further develops into an oocyst and eventually into new sporozoites, which migrate to the insect's salivary glands to infect a new vertebrate host.

[0004] Malaria symptoms are caused by blood-stage parasites. In contrast, sporozoites are not associated with clinical symptoms, however, numbers in the host are low during the sporozoite and liver stages of the Plasmodium parasite life cycle, and their elimination can completely eliminate the infection. Therefore, the sporozoite and liver stages of the Plasmodium falciparum parasite represent key targets for current malaria prevention candidates, as interventions that successfully provide protection against these stages will be able to prevent both malaria infection and transmission. Vaccines or molecules that block sporozoites are therefore at the core of the development of malaria prevention efforts.

[0005] Plasmodium circumsporozoite protein (CSP) is a membrane-bound protein that is present only in the sporozoite stage of Plasmodium. CSP forms a dense shell on the surface of the parasite, and it has been hypothesized that CSP mediates many of the initial interactions between sporozoites and their two hosts (Menard, 2000, Microbes Infect. 2: 633-642; Sinnis and Nardin, 2002, Chem Immunol 80: 70-96). The structure and function of CSP are highly conserved between various strains of Plasmodium that infect humans, non-human primates, and rodents. The amino acid sequence of CSP contains an immunodominant central repeat region that differs between Plasmodium species (NANP repeat region in the case of Plasmodium falciparum). The repeat is flanked by two conserved motifs and a known cell adhesion motif, which is located at the C-terminus of a repeat sequence called type I thrombospondin repeat (TSR). These conserved motifs are involved in protein processing when the parasite travels from the mosquito to the mammalian vector. It is known that CSP plays a crucial role in the migration of sporozoites from the midgut wall of infected mosquitoes to the mosquito salivary glands. Additionally, CSP participates in hepatocyte binding in mammalian hosts, where the N-terminal CSP initially promotes parasite binding. On the surface of hepatocytes, proteolytic cleavage at the N-terminal region I exposes the adhesive C-terminal domain, thereby priming the parasite to invade the liver (Coppi et al., 2005, J Exp Med 201, 27-33).

[0006] Currently, the most advanced malaria vaccine candidate is RTS, S (RTS, S / AS01; trade name Mosquirix), which is a recombinant protein-based malaria vaccine. RTS, S is a hybrid protein particle formulated in a multi-component adjuvant called AS01. The RTS, S vaccine antigen consists of 19 NANP amino acid repeating units, followed by a complete C-terminal domain minus the GPI anchor of the CSP antigen, fused to the hepatitis B virus S protein. Multi-site clinical trials conducted in sub-Saharan Africa have shown that RTS, S confers moderate and short-term protection against clinical malaria (RTS, S Clinical Trials Partnership, 2015, Lancet. 386 (9988): 31-45).

[0007] Another factor complicating the development of malaria treatments and prevention is that, although recent progress has been made in identifying immune-related factors associated with antibody specificity and function, the nature of the mechanisms that confer robust immune protection has been difficult to identify (Suscovich et al., 2020, Sci Transl Med 12(553):eabb4757).

[0008] Recently, anti-malarial antibodies specific for Plasmodium falciparum CSP have been described (Tan et al., 2018, Nat Med 24(4):401-407; Kisalu et al., 2018, Nat Med 24(4):408-416; Wang et al., 2020, Immunity 13;53(4):733-744; Pholcharee et al., 2021, Nat Commun 16;12(1):1063). However, although these CSP-binding antibodies have been described as protective in mouse preclinical infection models, they all have a proposed mechanism of action in which sporozoites are immobilized in the skin or prevented from binding to and / or infecting target cells in the liver through a mechanism mediated solely by the antigen-binding Fab region of the antibody (e.g., CIS43, L9). It is increasingly recognized that antibody Fc-mediated effector mechanisms can play an important role in preventing infection. In addition, the use of amino acid replacements in the antibody Fc region to manipulate these mechanisms can significantly improve the overall protection level conferred by the antibody. Recently published studies on immune-related factors (Suscovich et al., 2020, Sci Transl Med 12(553):eabb4757) point out this role in malaria, and other studies have previously demonstrated this principle in the context of other infectious diseases (Hiatt et al., 2014, PNAS 111; 5992-5997; DiLillo et al., 2014, Nat Med 20(2)143-151).

[0009] In view of the above, it is an object of the present invention to overcome the limitations of prior art malaria treatments outlined above by providing not only antibodies with high affinity, but also antibodies that confer the benefit of other protective mechanisms and an improved safety profile as well as ease of development (given the inherent problems of administration in malaria endemic countries). Summary of the invention

[0010] In one aspect of the present invention, the present invention provides a Plasmodium falciparum antigen binding protein.

[0011] In one aspect, the present invention provides a Plasmodium falciparum circumsporozoite protein (CSP) antigen binding protein comprising:

[0012] The heavy chain (HC) sequence of SEQ ID NO:9; and

[0013] The light chain (LC) sequence of SEQ ID NO:10.

[0014] In one aspect, the present invention provides a nucleic acid sequence encoding the heavy chain of SEQ ID NO:9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO:10.

[0015] In one aspect, the present invention provides one or more expression vectors comprising a nucleic acid sequence encoding the heavy chain of SEQ ID NO:9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO:10.

[0016] In one aspect, the present invention provides a recombinant host cell comprising: a nucleic acid sequence encoding the heavy chain of SEQ ID NO: 9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO: 10, or one or more expression vectors comprising a nucleic acid sequence encoding the heavy chain of SEQ ID NO: 9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO: 10.

[0017] In one aspect, the present invention provides a method for producing a circumsporozoite protein (CSP) antigen binding protein according to the present invention as disclosed herein, the method comprising culturing a host cell under conditions suitable for expressing one or more nucleic acid sequences or one or more vectors according to the present invention as described herein, thereby producing a polypeptide comprising the CSP antigen binding protein.

[0018] In one aspect, the present invention provides a pharmaceutical composition comprising a CSP antigen binding protein according to the present invention as described herein and a pharmaceutically acceptable excipient.

[0019] In one aspect, the present invention provides a method for preventing malaria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CSP antigen binding protein according to the present invention as described herein or a pharmaceutical composition comprising a CSP antigen binding protein according to the present invention as described herein.

[0020] In one aspect, the CSP antigen binding protein according to the present invention as described herein or a pharmaceutical composition comprising a CSP antigen binding protein according to the present invention as described herein is for use in preventing malaria. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Diversity analysis of antibody hit panels.

[0022] Figure 2 : Heparin binding ELISA data on the hit detection panel of antibodies showed a series of molecules with higher heparin binding profiles than the negative control, including AB-000325, AB-000337, AB001446 and AB-001516, which were subsequently removed from further analysis. Antibodies were incubated on heparin-coated poly-L-lysine plates for two hours prior to detection with anti-F(ab)'2-HRP.

[0023] Figure 3 : Heparin binding ELISA data on a test panel of Fc variants of antibodies. Heparin ELISA binding data on a test panel of mAb A antibodies carrying different amino acid substitutions to confer half-life extension and improved effector function.

[0024] Figure 4 : Human serum stability on a panel of Fc variants of antibodies. mAb-A Fc variants did not show much difference in stability in human plasma, but different molecules have inherently different stabilities, i.e. mAb-A vs. mAb-B. In vitro human serum stability over 8 weeks using CSP coated plates for capture and sulfo-tagged anti-human IgG1 Fc mAb for antibody detection is shown.

[0025] Figure 5 : In vivo evaluation of the effects of Fc enhancement on antibody potency in the Plasmodium berghei model. Figure 5 a shows liver burden measured by bioluminescence at 44 h. Figure 5 b shows day 5 after challenge with sporozoites at a dose of 25 μg (percentage of infected RBCs). Figure 5 c shows day 8 after challenge with a sporozoite dose of 100 μg (percentage of infected RBCs). Figure 5d shows the pharmacokinetic blood concentrations of antibodies at 2, 24, 44 and 96 hours after sporozoite challenge.

[0026] Taking into account the differences in serum concentrations between each mAb, statistical analysis of 356-ALE-LS vs. 356-LS showed very small effects of Fc enhancement, with the probability of 356-ALE-LS being at least 1-fold more potent than 356-LS approaching or exceeding 90% (87.60% on day 2, 99.97% on day 3, and 94.96% on day 5). For three-fold changes, these probabilities dropped to 21.06% (day 2), 63.05% (day 3), and 5.55% (day 5).

[0027] Figure 6 In vitro evaluation of CSP-mediated activation of primary NK cells (isolated from both wild-type C57BL / 6 mice and mice transgenic for human Fcγ receptors). Recombinant CSP protein was coated onto microtiter plates, followed by incubation with anti-CSP antibodies, and then NK cells isolated from the spleen of wild-type C57BL / 6 mice or transgenic mice expressing human FcγRs were added and incubated for three hours. NK activation was assessed by CD107a staining.

[0028] Figure 7 Quantification of parasites in peripheral blood at day 3 after sporozoite challenge was measured by real-time PCR. Passive transfer (100 μg (left) and 25 μg (right)) of the indicated mAbs (EpoFix as isotype control, 356-LS, 356-ALE-LS, 356-LAGA-LS) was administered in WT C57BL / 6 and human FcγR mice 24 hours prior to challenge with 1500 sporozoites.

[0029] Figure 8 In vivo neutralization activity of 356-ALE-LS and L9-LS against P. falciparum in FRG-huHep. (A) Representative images of parasite liver burden measured by bioluminescence of transgenic P. falciparum parasites expressing luciferase. (B) Reduction in liver burden (total flux, photons / second) 6 days post-infection. (C) Protection data at day 8 after sporozoite challenge. (D) Serum antibody concentration (μg / mL) at the time of parasite challenge. DETAILED DESCRIPTION

[0030] In one aspect of the present invention, the present invention provides a Plasmodium falciparum circumsporozoite protein (CSP) antigen binding protein.

[0031] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5 and CDRL3 of SEQ ID NO: 6, and wherein the antigen binding protein is modified to have enhanced effector function and increased half-life.

[0032] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15 and CDRL3 of SEQ ID NO: 16, and wherein the antigen binding protein is modified to have enhanced effector function or increased half-life.

[0033] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15 and CDRL3 of SEQ ID NO: 16, and wherein the antigen binding protein is modified to have enhanced effector function and increased half-life.

[0034] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5 and CDRL3 of SEQ ID NO: 6, and wherein the antigen binding protein is modified to have enhanced effector function or increased half-life.

[0035] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15 and CDRL3 of SEQ ID NO: 16, and wherein the antigen binding protein is modified to have enhanced effector function or increased half-life.

[0036] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein, comprising: CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein is mutated at least one of the following positions or combinations of positions:

[0037] i) S239D

[0038] ii) I332E

[0039] iii) S239D / I332E

[0040] iv) S239D / I332E / A330L

[0041] v) G236A / I332E

[0042] vi) S239D / I332E / G236A

[0043] vii)G236A / S239D / I332E / A330L

[0044] viii)H268F / S324T / G236A / I332E

[0045] ix) S267E / H268F / S324T / G236A / I332E

[0046] x) S298A / E333A / K334A

[0047] xi) F243L / R292P / Y300L / P396L

[0048] xii)F243L / R292P / Y300L / V305I / P396L

[0049] xiii)L235V / F243L / R292P / Y300L / P396L

[0050] xiv)F243L / R292P / Y300L

[0051] xv)G236A / A330L / I332E

[0052] xvi)K228M / K334E

[0053] xvii)S239D / I332E / G236A / T250V / A287F

[0054] xviii)H268F / S324T / S239D / I332E

[0055] xix)T250V / A287F / G236A / A330L / I332E

[0056] xx)T250V / A287F

[0057] And wherein the antigen binding protein has enhanced effector function.

[0058] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein is mutated at least one of the following positions or combinations of positions:

[0059] i) S239D

[0060] ii) I332E

[0061] iii) S239D / I332E

[0062] iv) S239D / I332E / A330L

[0063] v) G236A / I332E

[0064] vi) S239D / I332E / G236A

[0065] vii)G236A / S239D / I332E / A330L

[0066] viii)H268F / S324T / G236A / I332E

[0067] ix) S267E / H268F / S324T / G236A / I332E

[0068] x) S298A / E333A / K334A

[0069] xi) F243L / R292P / Y300L / P396L

[0070] xii)F243L / R292P / Y300L / V305I / P396L

[0071] xiii)L235V / F243L / R292P / Y300L / P396L

[0072] xiv)F243L / R292P / Y300L

[0073] xv)G236A / A330L / I332E

[0074] xvi)K228M / K334E

[0075] xvii)S239D, I332E, G236A, T250V, A287F

[0076] xviii)H268F / S324T / S239D / I332E

[0077] xix)T250V / A287F / G236A / A330L / I332E

[0078] xx)T250V / A287F

[0079] And wherein the antigen binding protein has enhanced effector function.

[0080] It is known from studies of naturally infected human patients that antibody effector functions can develop over time and this is associated with improved protection. However, this only occurs after multiple mosquito bites of Plasmodium falciparum and may take many years to develop (Feng et al., 2021, Nat Comms 12: 1742). There are currently no direct-acting anti-malarial antibodies that have been shown to have Fc-mediated protection, and there are no direct-acting anti-malarial antibodies that have been further modified to improve Fc-mediated host effector cell mechanisms.

[0081] Since it is difficult to show cellular killing of parasites and parasite-infected cells by targeting CSP (which is not naturally expressed in human cells in the absence of infection), the inventors have shown improved anti-malarial activity by modifying the Fc region to show improved antibody-dependent NK cell activation (ADNKA) and antibody-dependent cellular phagocytosis (ADCP) effector activity, and this as a marker of increased antibody-mediated effector function or ADCC. Therefore, the use of mAbs with such improved effector function can provide this additional protective mechanism at the earliest possible stage, especially in young children, thereby enabling more effective prevention.

[0082] An additional advantage of the antigen-binding proteins of the present invention is the formation of immune complexes (ICs). When bound to infectious parasites, the antigen-binding proteins can participate in the host adaptive immune response by binding to antigen-presenting cells (particularly dendritic cells (DCs)) and initiating T-cell and B-cell immune responses targeting infectious antigens through subsequent antigen presentation. In this way, a "vaccine-like" response to infectious agents can be elicited, providing improved long-term immune protection. This will bring additional benefits to the above-mentioned short-term immune preventive direct-acting Fab and Fc effector function mechanisms. Fc-enhanced antigen-binding proteins, such as mAbs of the present invention, can have a more significant effect by providing improved targeting to antigen-presenting cells and thus improved participation in the adaptive host immune response.

[0083] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5 and CDRL3 of SEQ ID NO: 6, and wherein the antigen binding protein is modified to have an increased half-life.

[0084] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15 and CDRL3 of SEQ ID NO: 16, and wherein the antigen binding protein is modified to have an increased half-life.

[0085] As used herein, the term "increased half-life" refers to the increase in the time required for the serum concentration of an antigen binding protein to reach half of its original value relative to a wild-type antigen binding protein (particularly an IgG1 antibody without modifications to the Fc region) when measured in an FcRn binding assay.

[0086] Various mechanisms are described throughout to increase half-life and are considered aspects of the invention as described herein. In addition to those amino acid substitutions that improve ADNKA and ADCP, amino acid substitutions that extend serum half-life were added because it is desirable to maintain serum concentrations of malarial mAbs over extended periods to minimize antibody administration in malaria-endemic countries to cover seasonal malaria.

[0087] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein is mutated at one of the following position combinations:

[0088] M428L and N434S

[0089] M252Y, S254T and T256E

[0090] H433K and N434F

[0091] And wherein the antigen binding protein has an increased half-life.

[0092] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15, and CDRL3 of SEQ ID NO: 16, and wherein the antigen binding protein is mutated at one of the following position combinations:

[0093] M428L and N434S

[0094] M252Y, S254T and T256E

[0095] H433K and N434F

[0096] And wherein the antigen binding protein has an increased half-life.

[0097] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5 and CDRL3 of SEQ ID NO: 6, and wherein the antigen binding protein is modified to have enhanced effector function and increased half-life.

[0098] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15 and CDRL3 of SEQ ID NO: 16, and wherein the antigen binding protein is modified to have enhanced effector function and increased half-life.

[0099] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5 and CDRL3 of SEQ ID NO: 6, and wherein the antigen binding protein comprises mutations G236A, A330L, I332E, M428L and N434S.

[0100] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15 and CDRL3 of SEQ ID NO: 16, and wherein the antigen binding protein comprises mutations G236A, A330L, I332E, M428L and N434S.

[0101] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5 and CDRL3 of SEQ ID NO: 6, and wherein the antigen binding protein comprises mutations S239D, I332E, G236A, T250V, A287F, M428L and N434S.

[0102] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising: CDRH1 of SEQ ID NO: 11, CDRH2 of SEQ ID NO: 12, CDRH3 of SEQ ID NO: 13, CDRL1 of SEQ ID NO: 14, CDRL2 of SEQ ID NO: 15 and CDRL3 of SEQ ID NO: 16, and wherein the antigen binding protein comprises mutations S239D, I332E, G236A, T250V, A287F, M428L and N434S.

[0103] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising a variable heavy chain of SEQ ID NO: 7 and a variable light chain of SEQ ID NO: 8, and wherein the antigen binding protein has enhanced effector function and increased half-life.

[0104] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising a variable heavy chain of SEQ ID NO: 7 and a variable light chain of SEQ ID NO: 8, and wherein the antigen binding protein is mutated at positions G236A, A330L, I332E, M428L and N434S. In one aspect, the antigen binding protein has enhanced effector function and increased half-life.

[0105] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising a variable heavy chain of SEQ ID NO: 7 and a variable light chain of SEQ ID NO: 8, and wherein the antigen binding protein is mutated at positions S239D, I332E, G236A, T250V, A287F, M428L and N434S. In one aspect, the antigen binding protein has enhanced effector function and increased half-life.

[0106] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising a variable heavy chain of SEQ ID NO: 17 and a variable light chain of SEQ ID NO: 18, and wherein the antigen binding protein has enhanced effector function and increased half-life.

[0107] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising a variable heavy chain of SEQ ID NO: 17 and a variable light chain of SEQ ID NO: 18, and wherein the antigen binding protein is mutated at positions G236A, A330L, I332E, M428L and N434S. In one aspect, the antigen binding protein has enhanced effector function and increased half-life.

[0108] In another aspect, the present invention provides a circumsporozoite protein (CSP) antigen binding protein comprising a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:10.

[0109] In one aspect, the present invention provides a nucleic acid sequence encoding the heavy chain of SEQ ID NO:9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO:10.

[0110] In one aspect, the present invention provides one or more expression vectors comprising a nucleic acid sequence encoding the heavy chain of SEQ ID NO:9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO:10.

[0111] In one aspect, the present invention provides a recombinant host cell comprising: a nucleic acid sequence encoding the heavy chain of SEQ ID NO: 9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO: 10, or one or more expression vectors comprising a nucleic acid sequence encoding the heavy chain of SEQ ID NO: 9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO: 10.

[0112] In one aspect, the present invention provides a method for producing a CSP antigen binding protein according to the present invention as disclosed herein, the method comprising culturing a host cell under conditions suitable for expressing one or more nucleic acid sequences or one or more vectors according to the present invention as described herein, thereby producing a polypeptide comprising the CSP antigen binding protein.

[0113] In one aspect, the present invention provides a pharmaceutical composition comprising a CSP antigen binding protein according to the present invention as described herein and a pharmaceutically acceptable excipient.

[0114] In one aspect, the present invention provides a method for preventing malaria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CSP antigen binding protein according to the present invention as described herein or a pharmaceutical composition comprising a CSP antigen binding protein according to the present invention as described herein.

[0115] In one aspect, the CSP antigen binding protein according to the present invention as described herein or a pharmaceutical composition comprising a CSP antigen binding protein according to the present invention as described herein is for use in preventing malaria.

[0116] In one aspect, the present invention provides use of a CSP binding protein according to the present invention as described herein or a pharmaceutical composition comprising a CSP binding protein according to the present invention as described herein in the preparation of a medicament for preventing malaria.

[0117] As used herein, the term "CSP binding protein" refers to antibodies and other protein constructs (such as domains) that are capable of binding to the circumsporozoite protein of Plasmodium falciparum. The terms "CSP binding protein" and "CSP antigen binding protein" are used interchangeably herein.

[0118] The term "antibody" is used in the broadest sense herein to refer to molecules with immunoglobulin-like domains (e.g., IgG, IgM, IgA, IgD or IgE), and includes monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, multispecific antibodies, including bispecific antibodies and heteroconjugate antibodies; antigen-binding antibody fragments, Fab, F(ab')2, Fv, disulfide-linked Fv, single-chain Fv, disulfide-linked scFv, diabodies, TANDABS, etc., and modified versions of any of the foregoing (for an overview of alternative antibody formats, see Holliger and Hudson, 2005, Nature Biotechnology, 23(9): 1126-1136).

[0119] The terms "whole antibody", "complete antibody" or "intact antibody" are used interchangeably herein and refer to a heterotetrameric glycoprotein with a molecular weight of approximately 150,000 Daltons. A complete antibody consists of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. The H2L2 structure folds to form three functional domains, which include two antigen-binding fragments (called "Fab" fragments) and an "Fc" crystallizable fragment. The Fab fragment consists of the following: a variable domain at the amino terminus, either variable heavy (VH) or variable light (VL), and a constant domain at the carboxyl terminus, namely CH1 (heavy) and CL (light). The Fc fragment consists of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc can elicit effector functions by binding to receptors on immune cells or by binding to C1q, the first component of the classical complement pathway. The five classes of antibodies, IgM, IgA, IgG, IgE, and IgD, are defined by different heavy chain amino acid sequences, called μ, α, γ, ε, and δ, respectively, and each heavy chain can be paired with either a κ or λ light chain. Most antibodies in serum belong to the IgG class, and there are four isotypes of human IgG (IgG1, IgG2, IgG3, and IgG4), whose sequences differ primarily in their hinge regions.

[0120] In one aspect of the invention, the CSP antigen binding protein is an antibody, as described herein. In another aspect, it is a monoclonal antibody. In another aspect, it is an IgG1 antibody.

[0121] Fully human antibodies can be obtained using a variety of methods, such as using a library based on yeast or a transgenic animal (e.g., mouse) that can produce a human antibody repertoire. Yeast that presents human antibodies that are bound to an antigen of interest on its surface can be selected using a method based on FACS (fluorescence activated cell sorting) or by capturing on a bead using a labeled antigen. Transgenic animals that have been modified to express human immunoglobulin genes can be immunized with an antigen of interest and use B cell sorting techniques to separate antigen-specific human antibodies. The desired properties of the human antibodies produced using these techniques can then be characterized, such as affinity, developability, and selectivity.

[0122] Alternative antibody formats include alternative scaffolds in which one or more CDRs of the antigen binding protein can be arranged on a suitable non-immunoglobulin protein scaffold or framework, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005 / 0053973, 2005 / 0089932, 2005 / 0164301) or an EGF domain.

[0123] Antigen binding site refers to the site on an antigen binding protein that is capable of specific binding to an antigen, which may be a single variable domain, or may be a paired VH / VL domain as may be found on a standard antibody. Single-chain Fv (ScFv) domains may also provide an antigen binding site.

[0124] Affinity, also referred to as binding affinity, refers to the strength of binding at a single interaction site, i.e., the strength of binding of a molecule (e.g., an antigen-binding protein of the present invention) to another molecule (e.g., its target antigen) at a single binding site. The binding affinity of an antigen-binding protein to its target can be determined by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., surface plasmon resonance (SPR) analysis). For example, the SPR method described in Example 1 can be used to measure binding affinity.

[0125] In one aspect, the CSP antigen binding protein binds with an affinity of at least 2.5 nM when measured by surface plasmon resonance (SPR).

[0126] In one aspect, the equilibrium dissociation constant (KD) of the antigen-binding protein interaction is 3nM or less. For example, the antigen-binding protein interaction is 2.5nM or less, or for example, 2nM or less, for example, 1nM or less, or for example, 100pM or less, or for example, 70pM or less. In one aspect, the antigen-binding protein interaction is 45pM or less. It will be appreciated by the technician that the smaller the KD value, the stronger the combination. In one aspect, the antigen-binding protein interaction is 10pM to 3nM or for example, 1nM to 2.5nM.

[0127] As used throughout this specification, the term "neutralize" means that the biological activity of a CSP is reduced in vitro or in vivo in the presence of an antigen binding protein as described herein, compared to the activity of the CSP in the absence of the antigen binding protein. Neutralization may be due to one or more of the following: blocking the binding of the CSP to its receptor, preventing the CSP from activating its receptor, preventing the proteolysis of the CSP, downregulating the CSP or its receptor, or affecting the effector function neutralization capacity of the anti-CSP binding protein.

[0128] CDR is defined as the complementary determining region amino acid sequence of antigen binding protein. These are the hypervariable regions of immunoglobulin heavy chain and light chain. The variable part of immunoglobulin has three heavy chain CDR and three light chain CDR (or CDR region). Therefore, as used herein, CDR refers to all three heavy chain CDR, all three light chain CDR, all heavy chain CDR and light chain CDR, or at least two CDR.

[0129] Throughout this specification, the amino acid residues in the variable domain sequence and the variable domain region within the full-length antigen binding sequence (e.g., within the antibody heavy chain sequence or the antibody light chain sequence) are numbered according to the Kabat numbering convention. Similarly, the terms CDR, CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, CDRH3 used in the examples follow the Kabat numbering convention. For more information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987).

[0130] It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, such as those set forth in Chothia et al., 1989, Nature, 342:877-883. The structure and protein folding of the antigen binding protein may mean that other residues are considered part of the CDR sequence, and the skilled artisan should understand this to be the case.

[0131] Other numbering conventions for CDR sequences available to the skilled artisan include the AbM (University of Bath) method and the contact (University College London) method.

[0132] Table A below represents one definition for each CDR or binding unit using each numbering convention. The variable domain amino acid sequences are numbered using the Kabat numbering scheme in Table A. It should be noted that some of these CDR definitions may vary depending on the individual publication used.

[0133] Table A

[0134]

[0135] The CDRs may be modified by at least one amino acid substitution, deletion or addition, wherein the variant antigen binding protein substantially retains the biological characteristics of the unmodified protein.

[0136] It will be appreciated that each of CDR H1, H2, H3, L1, L2, L3 can be modified individually or in any arrangement or combination, in combination with any other CDR. In one embodiment, a CDR is modified by substitution, deletion or addition of up to 3 amino acids (e.g., 1 or 2 amino acids, e.g., 1 amino acid). Typically, the modification is a substitution, particularly a conservative substitution, e.g., as shown in Table B below.

[0137] Table B:

[0138]

[0139]

[0140] For example, in a variant CDR, flanking residues comprising a CDR that is part of one or more alternative definitions (eg, Kabat or Chothia) may be replaced with conserved amino acid residues.

[0141] Such antigen binding proteins comprising variant CDRs as described above may be referred to herein as functional CDR variants.

[0142] The numbering used for mutations or combinations of mutations is according to standard antibody numbering and will be understood by those skilled in the art.

[0143] Neutralization can be determined or measured using one or more assays known to the skilled artisan or as described herein.

[0144] The percent identity or % identity between a query nucleic acid sequence and a subject nucleic acid sequence is an identity value expressed as a percentage, which is calculated over the full length of the query sequence using a suitable algorithm (e.g., BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign) after pairwise global sequence alignments have been performed using a suitable algorithm (e.g., Needleman-Wunsch or GenePAST / KERR) or software (e.g., DNASTAR Lasergene or GenePAST / KERR). Importantly, the query nucleic acid sequence may be described by a nucleic acid sequence disclosed herein (particularly in one or more of the claims).

[0145] The percent identity or % identity between the query amino acid sequence and the target amino acid sequence is calculated over the full length of the query sequence using a suitable algorithm (e.g., BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle, or EMBOSS infoalign) after a pairwise global sequence alignment has been performed using a suitable algorithm (e.g., Needleman-Wunsch or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle, or EMBOSS infoalign). Importantly, the query amino acid sequence can be described by an amino acid sequence disclosed herein (particularly in one or more of the claims).

[0146] The query sequence can be 100% identical to the target sequence, or compared to the target sequence, the query sequence can include at most a certain integer number of amino acids or nucleotide changes so that the identity % is less than 100%. For example, the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the target sequence. In the case of nucleic acid sequences, such changes include the deletion, replacement or insertion of at least one nucleotide residue, wherein the change can occur at the 5'- or 3'-terminal position of the query sequence or any position between these terminal positions, scattered in the nucleotide residues in the query sequence alone or in one or more adjacent groups within the query sequence. In the case of amino acid sequences, such changes include at least one amino acid residue deletion, replacement (including conservative and non-conservative replacement) or insertion, wherein the change can occur at the amino or carboxyl terminal position of the query sequence or any position between these terminal positions, scattered in the amino acid residues in the query sequence alone or in one or more adjacent groups within the query sequence.

[0147] For antibody sequences, the % identity can be determined over the full length of the query sequence (including CDRs). Alternatively, the % identity can exclude one or more or all CDRs, such as all CDRs are 100% identical to the target sequence and the % identity variation is in the remainder of the query sequence (e.g., framework sequence), such that the CDR sequence is fixed and complete. The variant sequence substantially retains the biological characteristics of the unmodified protein.

[0148] As used herein, the term "effector function" refers to one or more antibody-mediated effects, including antibody-dependent NK activation (ADNKA), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated complement activation (including complement-dependent cytotoxicity (CDC), antibody-dependent complement-mediated cell lysis (ADCML)), and Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP) by effector cells (including macrophages and neutrophils).

[0149] In a further aspect, "effector function" as used throughout this specification is intended to refer to one or more antibody-dependent NK activation (ADNKA), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cellular phagocytosis (ADCP). In a further aspect, the effector function is ADCC.

[0150] The interaction between the Fc region of an antigen binding protein or antibody and various Fc receptors (FcR) and complement factors (including FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcRn, C1q and type II Fc receptor) is believed to mediate the effector function of the antigen binding protein or antibody. Significant biological effects can be the result of effector function. Generally, the ability to mediate effector function requires the antigen binding protein or antibody to bind to the antigen, and not all antigen binding proteins or antibodies will mediate every effector function.

[0151] Effector function can be assessed in a variety of ways, including, for example, assessing the ADCC effector function of antibodies coated to target cells (mediated by natural killer (NK) cells via FcγRIIIa) or ADCP effector function (mediated by monocytes / macrophages or neutrophils via FcγRIIa), or assessing the CDC effector function of antibodies coated to target cells (mediated by the complement cascade via C1q). For example, the ADCC effector function of the antigen binding proteins of the invention can be assessed in a natural killer cell activation assay. Examples of such assays can be found in Shields et al., 2001, J Biol Chem, 276: 6591-6604; Lazar et al., 2006, PNAS, 103; 4005-4010.

[0152] Throughout the present specification, amino acid residues in the Fc region, in the antibody sequence or in the full-length antigen binding protein sequence are numbered according to the EU index numbering convention.

[0153] Human IgG1 constant regions containing specific mutations have been shown to enhance binding to Fc receptors. In some cases, these mutations have also been shown to enhance effector functions, such as ADCC, ADCP, and CDC, as described below. The antigen binding proteins of the present invention may include any of the following mutations.

[0154] Enhanced CDC: Fc engineering can be used to enhance complement-based effector functions. For example (refer to IgG1), K326W / E333S; S267E / H268F / S324T and IgG1 / IgG3 cross-subclass can increase C1q binding; single E345R and triple E345R / E430G / S440Y mutations lead to preformed IgG hexamer (Diebolder et al., Science 2014; 343: 1260-1263; Wang et al., 2018, Protein Cell, 9(1): 63–73).

[0155] Enhanced ADCC: Fc engineering can be used to enhance ADCC. For example (refer to IgG1), F243L / R292P / Y300L / V305I / P396L; S239D / I332E; and S298A / E333A / K334A increase FcγRIIIa binding; S239D / I332E / A330L increases FcγRIIIa binding and reduces FcγRIIb binding; an asymmetric Fc containing L234Y / L235Q / G236W / S239M / H268D / D270E / S298A mutations in one heavy chain and D270E / K326D / A330M / K334E in the opposing heavy chain increases binding to FcγRIIIa F158 (lower affinity allele) and FcγRIIIa V158 (a higher affinity allele) was found to have an increased affinity for β-catenin, while having no increased binding affinity for inhibitory FcγRIIb (Wang et al., 2018, Protein Cell, 9(1):63–7).

[0156] Enhanced ADCP: Fc engineering can be used to enhance ADCP. For example (reference IgG1), G236A / S239D / I332E increases FcγRIIa binding and increases FcγRIIIa binding (Richards et al., Mol Cancer Ther, 2008, 7:2517-2527). G236A / S239D / I332E improves binding to FcγRIIa, improves FcγRIIa / FcγRIIb binding ratio (activation / inhibition ratio), and enhances phagocytosis of antibody-coated target cells by macrophages (Wang et al., 2018, Protein Cell, 9(1):63–7).

[0157] Glycosylation

[0158] Antigen-binding proteins of the present invention can comprise the heavy chain constant region with the glycosylation profile of change, make this antigen-binding proteins have the effector function of enhancement, for example enhanced ADCC, enhanced CDC, enhanced ADCP or its combination.Examples of suitable methods for producing antigen-binding proteins with the glycosylation profile of change are described in WO2003011878, WO2006014679 and EP1229125, all of which methods can be applied to antigen-binding proteins of the present invention.

[0159] The lack of the innermost α1,6 fucose residue on the Fc glycan moiety at N297 of IgG1 antibodies enhances affinity for FcγRIIIA. Therefore, defucosylated or hypofucosylated monoclonal antibodies may have enhanced therapeutic efficacy (Shields et al., J Biol Chem, 2002, 277(30):26733-40; Monnet et al., 2014, mAbs, 6(2):422-436).

[0160] Complegent

[0161] In one embodiment of the present invention, the present invention provides an antigen-binding protein comprising a chimeric heavy chain constant region. In one embodiment, the antigen-binding protein comprises an IgG1 / IgG3 chimeric heavy chain constant region, so that the antigen-binding protein has enhanced CDC. For example, the chimeric antigen-binding protein of the present invention may comprise at least one CH2 domain from IgG3. In such an embodiment, the antigen-binding protein comprises a CH2 domain from IgG3, or both CH2 domains may be from IgG3. In one embodiment, the chimeric antigen-binding protein comprises an IgG1 CH1 domain, an IgG3 CH2 domain, and an IgG3 CH3 domain. In one embodiment, the chimeric antigen-binding protein comprises an IgG1 CH1 domain, an IgG3 CH2 domain, and an IgG3 CH3 domain (but position 435 is H (histidine)).

[0162] In one embodiment, the antigen binding protein comprises an IgG1 CH1 domain and at least one CH2 domain from IgG3. In one embodiment, the chimeric antigen binding protein comprises the following residues in the IgG1 CH1 domain and the CH2 domain corresponding to IgG3 residues: 274Q, 276K, 296F, 300F, and 339T. In one embodiment, the chimeric antigen binding protein also comprises 356E corresponding to IgG3 residues in the CH3 domain. In one embodiment, the antigen binding protein also comprises one or more of the following residues corresponding to IgG3 residues in the CH3 domain: 358M, 384S, 392N, 397M, 422I, 435R, and 436F.

[0163] Such methods for producing antigen-binding proteins with chimeric heavy chain constant regions can be used, for example, available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kirin Co., Ltd. COMPLEGENT technology systems are carried out. The COMPLEGENT system includes a recombinant host cell comprising an expression vector, in which a nucleic acid sequence encoding a chimeric Fc region with amino acid residues in IgG1 and IgG3 Fc regions is expressed, to produce antigen-binding proteins with enhanced CDC activity, i.e., an increase in CDC activity relative to antigen-binding proteins that are otherwise identical but lack such chimeric Fc regions, as described in WO2007011041 and US20070148165, each of which is incorporated herein by reference. In an alternative embodiment, CDC activity can be increased by introducing sequence-specific mutations into the Fc region of IgG chains. Those of ordinary skill in the art will also recognize other suitable systems.

[0164] Potelligent

[0165] Such methods for producing antigen binding proteins can be performed using, for example, the POTELLIGENT technology system available from BioWa, Inc. (Princeton, NJ), wherein CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with enhanced ADCC activity, which is increased relative to the same monoclonal antibody produced in cells with a functional FUT8 gene, as described in US7214775, US6946292, WO0061739 and WO0231240, all of which are incorporated herein by reference. Those of ordinary skill in the art will also recognize other suitable systems.

[0166] In one embodiment of the invention, the antigen binding protein is produced in a host cell in which the FUT8 gene has been inactivated. In one embodiment of the invention, the antigen binding protein is produced in a - / - FUT8 host cell. In one embodiment of the invention, the antigen binding protein is defucosylated at Asn297 (IgG1).

[0167] It will be apparent to those skilled in the art that such modifications can be used not only alone, but also in combination with one another to further enhance effector function.

[0168] The half-life of any antigen binding protein refers to the time required for the serum concentration of the antigen binding protein to reach half of its original value. The serum half-life of a protein can be measured by pharmacokinetic studies according to the methods described in Kim et al., 1994, Eur J Immunol 24:542-548. According to this method, a radiolabeled protein is injected intravenously into mice, and its plasma concentration is regularly measured over time, for example, from about 3 minutes to about 72 hours after injection. Other methods for pharmacokinetic analysis and determination of molecular half-life will be familiar to those skilled in the art.

[0169] It is reported that the long half-life of IgG antibodies depends on their binding to FcRn. Therefore, by engineering constant regions, the replacement of increasing the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependence of interacting with the target has been extensively studied (Ghetie et al., 1997, Nature Biotech, 15: 637-640; Hinton et al., 2004, J Biol Chem, 279: 6213-6216; Dall'Acqua et al., 2002, J Immunol 169 (9): 5171-5180). The in vivo half-life of the antigen-binding proteins of the present invention can be changed by modifying the heavy chain constant domain or the FcRn binding domain therein.

[0170] In adult mammals, FcRn (also known as neonatal Fc receptor) plays a key role in maintaining serum antibody levels by acting as a protective receptor that binds and rescues antibodies of the IgG isotype from degradation. IgG molecules are endocytosed by endothelial cells and, if they bind to FcRn, are recycled out of the cell back into the circulation. In contrast, IgG molecules that enter the cell and do not bind to FcRn are targeted to the lysosomal pathway, where they are degraded.

[0171] FcRn is believed to be involved in antibody clearance and transcytosis in tissues (see Junghans, 1997, Immunol Res, 16: 29-57 and Ghetie and Ward, 2000, Annu Rev Immunol 18: 739-766). Human IgG1 residues that have been determined to directly interact with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435. Mutations in any of these positions can increase the serum half-life of the antigen-binding protein of the present invention and / or alter the effector properties.

[0172] The antigen-binding proteins of the present invention may have amino acid modifications that increase the affinity of the constant domain or its fragment to FcRn. Increasing the half-life (i.e., serum half-life) of therapeutic and diagnostic IgG antibodies and other bioactive molecules has many benefits, including reducing the dosage and / or frequency of these molecules. In one embodiment, the antigen-binding proteins of the present invention comprise all or a portion (FcRn binding portion) of an IgG constant domain having one or more of the following amino acid modifications.

[0173] For example, referring to IgG1, M252Y / S254T / T256E (commonly referred to as YTE mutations) and M428L / N434S (commonly referred to as LS mutations) increase FcRn binding at pH 6.0 (Wang et al., 2018, Protein Cell, 9(1):63–73).

[0174] The half-life can also be increased by T250Q / M428L, V259I / V308F / M428L, N434A and T307A / E380A / N434A mutations (with reference to IgG1 and Kabat numbering) (Monnet et al., 2014, Mabs, 6(2):422-436).

[0175] Half-life and FcRn binding can also be extended by introducing H433K and N434F mutations (often referred to as HN or NHance mutations) (refer to IgG1) (WO2006 / 130834).

[0176] WO00 / 42072 discloses a polypeptide comprising a variant Fc region with altered FcRn binding affinity, the polypeptide comprising an amino acid modification at any one or more of the following amino acid positions: 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, mAb A, 340, 356, 360, 362, 376, 378, 380, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and 447 of the Fc region (EU index numbering).

[0177] WO02 / 060919 discloses a modified IgG comprising an IgG constant domain comprising one or more amino acid modifications relative to a wild-type IgG constant domain, wherein the modified IgG has an increased half-life compared to an IgG having a wild-type IgG constant domain, and wherein the one or more amino acid modifications are located at one or more of positions 251, 253, 255, 285-290, 308-314, 385-389 and 428-435.

[0178] Shields et al., 2001, J Biol Chem, 276:6591-6604 used alanine scanning mutagenesis to change residues in the Fc region of human IgG1 antibodies and then evaluated binding to human FcRn. Positions that effectively eliminated binding to FcRn when changed to alanine included I253, S254, H435, and Y436. Other positions showed less obvious decreases in binding, as follows: E233-G236, R255, K288, L309, S415, and H433. When changed to alanine, several amino acid positions showed improved FcRn binding; among them, it is worth noting that P238, T256, E272, V305, T307, Q311, D312, K317, D376, E380, E382, S424, and N434. Many other amino acid positions showed either slight improvements in FcRn binding (D265, N286, V303, K360, Q362, and A378) or no changes (S239, K246, K248, D249, M252, E258, T260, S267, H268, S269, D270, K274, N276, Y278, D280, V282, E283, H285, T289, K290, R292, E293, E294, Q295, Y296, N297, S298, R301, N315, E318, K320, K322, S324, K327, 421, V422, E430, T437, K439, S440, S442, S444 and K447).

[0179] With regard to improved FcRn binding, the most pronounced effect was seen with the combined variants. At pH 6.0, the E380A / N434A variant exhibited more than 8-fold better binding to FcRn relative to native IgG1, compared to 2-fold for E380A and 3.5-fold for N434A. Addition of T307A to it resulted in a 12-fold improvement in binding relative to native IgG1. In one embodiment, an antigen binding protein of the invention comprises the E380A / N434A mutations and has increased binding to FcRn.

[0180] Dall'Acqua et al., 2002, J Immunol 169(9):5171-5180 describe random mutagenesis and screening of a human IgG1 hinge-Fc fragment phage display library against mouse FcRn. They disclose random mutagenesis of positions 251, 252, 254-256, 308, 309, 311, 312, 314, 385-387, 389, 428, 433, 434, and 436. When the residues (M252, S254, T256, H433, N434 and Y436) located in the band across the Fc-FcRn interface are replaced, and to a lesser extent, when the residues (such as V308, L309, Q311, G385, Q386, P387 and N389) around the replacement, a significant improvement in the stability of the IgG1-human FcRn complex occurs. The variant with the highest affinity for human FcRn was obtained by combining M252Y / S254T / T256E (YTE) and H433K / N434F / Y436H mutations, and showed a 57-fold increase in affinity relative to wild-type IgG1. Compared with wild-type IgG1, the in vivo behavior of this mutant human IgG1 showed a nearly 4-fold increase in serum half-life in cynomolgus monkeys.

[0181] Thus, the present invention provides an antigen binding protein with optimized binding to FcRn. In a preferred embodiment, the antigen binding protein comprises at least one amino acid modification in the Fc region of the antigen binding protein, wherein the modification is at an amino acid position selected from the group consisting of: 226, 227, 228, 230, 231, 233, 234, 236, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 287, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, ​​384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447.

[0182] In addition, various publications describe methods for obtaining physiologically active molecules with modified half-lives by introducing FcRn-binding polypeptides into the molecules (WO97 / 43316, US5869046, US5747035, WO96 / 32478 and WO91 / 14438), or by fusing the molecules with antibodies that retain FcRn binding affinity but have greatly reduced affinity for other Fc receptors (WO99 / 43713) or with the FcRn-binding domain of an antibody (WO00 / 09560, US4703039).

[0183] Fc variants with enhanced FcRn affinity were identified in a screen at pH 6.0 to improve both the cytotoxicity and half-life of the antibody. The selected IgG variants can be produced as low-fucosylated molecules. The resulting variants showed increased serum persistence in hFcRn mice, as well as conservative enhanced ADCC (Monnet et al., 2014, mAbs, 6(2): 422-436). Exemplary variants include (with reference to IgG1 and Kabat numbering): P230T / V303A / K322R / N389T / F404L / N434S; P228R / N434S; Q311R / K334R / Q342E / N434Y; C226G / Q386R / N434Y; T307P / N389T / N434Y; P230S / N434S; P230T / V305A / T307A / A378V / L398P / N434S; P23OT / P387S / N434S; P230Q / E269D / N434S; N276S / A378V / N434S ;T307A / N315D / A330V / 382V / N389T / N434Y ; T256N / A378V / S383N / N434Y; N315D / A330V / N361D / A387V / N434Y; V259I / N315D / M428L / N434Y; P230S / N315D / M428L / N434Y; F241L / V264E / T307P / A378V / H433R; T250A / N389K / N434Y; V305A / N315D / A330V / P395A / N434Y; V264E / Q386R / P396L / N434S / K439R; E294del / T307P / N434Y (where "del" indicates deletion).

[0184] Although substitutions in the constant region can significantly improve the function of IgG antibodies, substitutions in strictly conserved constant regions have the risk of being immunogenic in humans (De Groot and Martin, 2009, Clin Immunol 131: 189-201), whereas substitutions in highly divergent variable region sequences may be less immunogenic. Reports on variable regions include engineering CDR residues to improve binding affinity to antigen (Rothe et al., 2006, Expert Opin Bioi Ther 6:177-187; Bostrom et al., 2009, Methods Mol Biol 525:353-376; Thie et al., Methods Mol Biol 525:309-322, 2009) and engineering CDR and framework residues to improve stability (Worn and Pluckthun, 2001, J Mol Biol 305:989-1010; Ewert et al., 2004, Methods 34:184-199) and reduce the risk of immunogenicity (De Groot and Martin, 2009, Clin Immunol 131:189-201; Jones et al., 2009, Methods Mol Biol 525:405-423, xiv). As reported, improved affinity to antigens can be achieved by affinity maturation using phage or ribosome display of random libraries.

[0185] Improved stability can be reasonably obtained by sequence-based and structure-based rational design. Reduced immunogenicity risk (deimmunization) can be achieved by various humanization methods and removal of potential T cell epitopes, which can be predicted using computer simulation (in silico) technology or estimated by in vitro assay. In addition, variable regions have been engineered to lower pI. Although FcRn binds fairly, compared with wild-type antibodies, it is observed that the half-life of these antibodies is longer. Engineering or selecting antibodies with pH-dependent antigen binding can be used to modify antibodies and / or antigen half-life, for example, if antigen-mediated clearance mechanisms normally degrade antibodies when bound to antigens, then IgG2 antibody half-life can be shortened. Similarly, antigen: antibody complexes can extend the half-life by protecting the antigen from typical degradation processes, or shorten the half-life by antibody-mediated degradation (target-mediated drug disposal), thereby affecting the half-life of the antigen. One embodiment relates to antibodies that have a higher affinity for an antigen at pH 7.4 compared to the endosomal pH (i.e., pH 5.5-6.0), such that the KD ratio at pH 5.5 / pH 7.4 or pH 6.0 / pH 7.4 is 2 or greater. For example, to enhance the pharmacokinetic (PK) and pharmacodynamic (PD) properties of an antibody, pH sensitivity can be engineered into the antibody by introducing histidine residues into the CDRs.

[0186] The antigen binding proteins as described herein can be incorporated into pharmaceutical compositions for preventing or treating human diseases as described herein, in particular for preventing malaria. In one embodiment, the pharmaceutical composition comprises a combination of the antigen binding protein and one or more pharmaceutically acceptable carriers and / or excipients.

[0187] In a further aspect, provided herein is a pharmaceutical composition for administering the CSP antigen binding protein of the invention to a mammalian subject (preferably a human) at risk of malaria in an amount and schedule sufficient to prevent Plasmodium infection. In another aspect, the subject resides in a malaria-endemic area of ​​sub-Saharan Africa. In one such aspect, the human subject is a child under 5 years of age. In another aspect, the subject is a pregnant woman.

[0188] Such compositions may comprise a CSP antigen binding protein as described herein, or a polynucleotide encoding the antigen binding protein, and a pharmaceutically acceptable diluent or carrier. In some embodiments, the polynucleotide encoding the antigen binding protein may be contained in a plasmid vector or a viral vector for delivery. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the antigen binding protein. As used herein, a therapeutically effective dose or a therapeutically effective amount refers to an amount sufficient to prevent, cure, or at least partially inhibit malaria or malaria symptoms. A therapeutically effective dose can be determined by monitoring the patient's response to therapy. Typical benchmarks indicating a therapeutically effective dose include alleviating or preventing the patient's malaria symptoms, including, for example, a reduction in the number of parasites.

[0189] In one aspect, prevention of malaria is complete prevention of development of liver stage disease and complete absence of blood stage disease.

[0190] In one aspect, prevention of malaria is measured as a delay in the time to detection of the parasite in the blood of a relevant subject, such as a child or pregnant woman in a malaria-endemic country, such as in sub-Saharan Africa.

[0191] In one aspect, antigen-binding proteins according to the present invention provide a reduced risk of malaria infection. In clinical trials, the risk of infection is measured as the level of protection achieved to a patient population compared to a placebo. For example, analysis can be based on the time of first infection with Plasmodium falciparum in a 24-week period. The average time of the first infection of a patient population using a placebo is much earlier than the group treated with mAb. In another example, the ratio of participants without Plasmodium falciparum infection was measured compared to a placebo in a 24-week period. In one aspect, the reduction in risk of infection after the calculated administration is at least about 20%, 30%, 40%, 50%, for example 60%, such as 65%.

[0192] In one aspect, antigen binding proteins according to the invention provide a reduced risk of clinical symptoms of malaria. In one aspect, after receiving, for example, at least one administration at a 3 month study interval, the risk of clinical disease can be reduced by at least about 20%, 30%, 40%, 50%, for example 60%, such as about 65%.

[0193] In one aspect, the malaria is non-severe malaria. In another aspect, the malaria is severe.

[0194] In one aspect, the antigen binding protein provides a reduced risk of developing asexual parasitosis of Plasmodium falciparum in a subject despite the presence of a fever above 37.5°C or a history of fever within 24 hours.

[0195] In one aspect, the reduced risk of malaria infection and the reduced risk of developing clinical symptoms of malaria are assessed over a period of 3 months following administration of the antigen binding protein.

[0196] In one aspect, the CSP binding protein provides protection for a period of at least 3 months following administration.

[0197] Clinical malaria is defined herein as the presence of malarial parasites in the blood. In a further example, clinical malaria may be defined as a fever greater than or equal to 37.5°C and asexual parasitosis with 500 or more Plasmodium falciparum per μL of blood (eg, with a sensitivity and specificity greater than 90%).

[0198] The definition of severe malaria can, for example, include the presence of one or more of the following: severe malarial anemia (PCV <15%), cerebral malaria (Blantyre Coma Scale <2), or severe illness of other body systems, which can include multiple seizures (two or more generalized convulsions in the previous 24 hours), collapse (defined as inability to sit independently), hypoglycemia (<2.2 mmol / dL or <40 mg / dL), clinically suspected acidosis, or circulatory collapse.

[0199] The effective amount will depend on factors such as age, body weight, route of administration, etc. Single or multiple administrations of the antibody will depend on the dose and frequency as required and tolerated by the patient. In some embodiments, the antibody is administered in a time frame to prevent hepatocyte infection during the pre-erythrocytic stage of infection.

[0200] The effective amount will depend on the severity of the disease and the patient's overall health, including other factors such as age, weight, sex, route of administration, etc. Single or multiple administrations of the antibody will depend on the dose and frequency as required and tolerated by the patient. In some embodiments, the antibody is administered during the pre-erythrocytic stage of infection, or within a time frame to prevent or reduce hepatocyte infection.

[0201] In one aspect, the antigen binding protein is administered at least once prior to the transmission season, in another aspect, the antigen binding protein is administered at least twice prior to the transmission season, in another aspect, the antigen binding protein is administered at least three times.

[0202] The pharmaceutical composition can be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, and intraportal). In one embodiment, the composition is suitable for intramuscular administration or subcutaneous administration. In one aspect, the administration is intramuscular. In one embodiment, the administration is subcutaneous. The pharmaceutical composition can be suitable for topical administration (which includes, but is not limited to, epicutaneous, inhaled, intranasal or ocular administration) or enteral administration (which includes, but is not limited to, oral, vaginal or rectal administration).

[0203] In one aspect, the CSP antigen binding protein according to the invention as described herein or the pharmaceutical composition as described herein or a pharmaceutical composition comprising the CSP antigen binding protein as described herein is for intravenous, subcutaneous or intramuscular administration. The pharmaceutical composition may be contained in a kit containing the antigen binding protein and other drugs and / or instructions for use. For convenience, the kit may contain predetermined amounts of these reagents and instructions for use. The kit may also contain a device for administering the pharmaceutical composition. In a further aspect, the kit may contain a diagnostic agent for detecting an infection.

[0204] The terms "individual," "subject," and "patient" are used interchangeably herein. In one embodiment, the subject is an animal. In another embodiment, the subject is a mammal, such as a primate, e.g., a woodchuck or a monkey. In another embodiment, the subject is a human.

[0205] The antigen binding proteins described herein can also be used in preventive or therapeutic methods. The antigen binding proteins described herein are used in effective amounts for prophylactic or preventative treatment. As used herein, a therapeutically effective dose or therapeutically effective amount refers to an amount sufficient to prevent, cure, or at least partially inhibit malaria or malaria symptoms.

[0206] In one aspect, the antigen binding proteins of the invention as described herein will be administered to subjects in need thereof, in another aspect, these antigen binding proteins will be administered to patients under the age of 5. In another aspect, the antigen binding proteins of the invention as described herein will be administered to pregnant women.

[0207] In one aspect, the antigen binding protein provides protection against blood-stage Plasmodium falciparum infection for at least 1 month, or at least 3 months, or at least 5 months, or at least 8 months, or at least 12 months.

[0208] Antigen binding proteins can be prepared by any of a variety of conventional techniques. For example, antigen binding proteins can be purified from cells that naturally express them (e.g., antibodies can be purified from hybridomas that produce them) or produced in recombinant expression systems.

[0209] A variety of different expression systems and purification schemes can be used to generate antigen-binding proteins of the present invention. Generally, host cells are transformed with recombinant expression vectors encoding one or more desired antigen-binding proteins. The expression vector can be maintained or integrated into the host chromosome as an independent genetic element by the host, depending on the expression system. Many kinds of host cells can be used, including but not limited to prokaryotes (including Gram-negative or Gram-positive bacteria, such as Escherichia coli, Bacillus sp., Pseudomonas sp., Corynebacterium sp.); eukaryotes, including yeast (such as Saccharomyces cerevisiae, Pichia pastoris), fungi (such as Aspergillus sp.) or higher eukaryotes, including insect cells and mammalian cell lines (e.g., CHO, NSO, PER.C6, HEK293, HeLa).

[0210] The host cell may be an isolated host cell. The host cell is not typically part of a multicellular organism (e.g., a plant or an animal). The host cell may be a non-human host cell.

[0211] Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian host cells are known in the art.

[0212] These cells can be cultured using various equipment (such as shake flasks, spinner flasks and bioreactors) under conditions that promote the expression of the antigen-binding protein. The polypeptide is recovered by conventional protein purification procedures. Protein purification procedures are usually composed of a series of unit operations (composed of various filtration and chromatographic processes developed for selective concentration and separation of the antigen-binding protein). The purified antigen-binding protein can be formulated in a pharmaceutically acceptable composition.

[0213] Table C - Sequence

[0214]

[0215]

[0216] Throughout the specification, for the avoidance of doubt, the nomenclature: 356-ALE-LS, AB-000356ALE LS and AB-356ALE_LS are used interchangeably and refer to the same antibody.

[0217] Example

[0218] Example 1 In vitro and in vivo characterization of a hit detection panel of malaria mAbs.

[0219] In the Phase 2 vaccine trial of RTS,S, a panel of 79 different antibody sequences originally derived from protected volunteers was selected for evaluation. The diversity of the antibody hit panel is shown in Figure 1 middle.

[0220] Because the developability and manufacturing potential of an antibody does not always align with optimal potency using in vitro potency models and in vivo preclinical disease models, this hit panel is first evaluated using an in vitro protein profiling assay to determine which antibodies will be further developed.

[0221] The remaining molecules in the hit detection panel were then subjected to small-scale (125 ml) antibody material generation, followed by protein analysis testing by size exclusion chromatography (SEC), mass spectrometry (MS), hydrophobic interaction chromatography (HIC), heparin binding ELISA, micro-differential scanning fluorimetry (nano-DSF) assay, and target binding affinity by surface plasmon resonance (SPR) using recombinant CSP. Antibodies that did not meet defined criteria were excluded from advancement, including those with less than the required purified material yield, those that did not meet the 95% monomer SEC threshold, those that showed binding above the positive control in the heparin binding ELISA assay ( Figure 2 ), antibodies that showed high retention time by HIC (Table 1) and those with low initial melting profiles (Table 2). In addition, SPR data showed several antibodies with no measurable binding or low binding>3.0 nM (Table 3).

[0222] The molecules that were not excluded (designated the lead panel) were subsequently tested for efficacy in the Plasmodium berghei mouse model of malaria sporozoite challenge.

[0223] Table 1. Hydrophobic interaction chromatography data on the hit detection panel of antibodies. All antibodies listed in this table have the M428L / N434S (LS) modification to extend the half-life.

[0224]

[0225]

[0226]

[0227]

[0228] Table 2 Nano-DSF data on a hit detection panel of antibodies, all with M428L / N434S (LS) modification to extend half-life.

[0229]

[0230]

[0231]

[0232]

[0233] Table 3 SPR data using recombinant CSP showing variable binding affinity. Note that the antibodies listed in this table all have M428L / N434S (LS) modifications to extend half-life.

[0234]

[0235]

[0236]

[0237] Example 2 In vivo potency assessment of the lead test panel of antibodies

[0238] The in vivo P. berghei mouse model of protection was used to evaluate the protective efficacy of the lead test panel. This model involves infection with C57BL / 6 mice (Lin et al., 2013, Methods Mol Biol, 923:507-22; Tewari et al., 2002, J Biol Chem, 277(49):47613-8) of a recombinant rodent P. berghei strain carrying P. falciparum CSP (which also carries a GFP transgenic marker to enable visualization of sporozoite infection of hepatocytes).

[0239] Since we have previously determined that a dose of 300 μg of antibody is fully protective in this mouse model, it was decided to select a dose of 200 μg in order to better evaluate the rank order of antibody efficacy. Animals (n=3) were administered with each mAb by the iv route 24 hours before the administration of 1500 sporozoites. The animals were then examined for liver infection by bioluminescence at 44 hours, and blood-stage disease was examined by PCR and flow cytometry to the 13th day on the 3rd day. The number of animals fully protected in each group was evaluated as the primary endpoint of the study. A summary of the results is summarized in Table 4. The results showed that there were great differences in the level of protection. At the end of the study, day 10, eight mAbs showed complete protection in only one of three animals (33% protection), and another eight mAbs conferred complete protection in two of three animals (66% protection), while only two mAbs (mAb 356 and 338) conferred 100% complete protection. Other mAbs did not show any protection at the end of the study. Interestingly, there was no clear relationship between antibody affinity and in vivo potency.The top ten most potent molecules were further triaged using a lower dose of 100 μg and the data are summarized in Table 5. Antibody 356 and antibody 338 were once again the most potent molecules.

[0240] Table 4 In vivo evaluation of hit panel antibodies in the mouse P. berghei model. All antibodies listed in this table have M428L / N434S (LS) modifications to extend half-life, except mAb A.

[0241]

[0242]

[0243]

[0244] Table 5 Top ten lead antibodies evaluated in vivo in the mouse P. berghei model at a dose of 100 μg. All antibodies listed in this table have the M428L / N434S (LS) modification to extend half-life, with the exception of mAb A in one of these studies (recorded in this table).

[0245]

[0246]

[0247] Example 3 Amino acid substitutions are selected as necessary to confer optimal Fc effector function.

[0248] When incorporating various amino acid substitutions into monoclonal antibodies with the intent to confer increased Fc effector function and half-life, there is a risk of compromising other attributes of the desired antibody drug profile, such as target binding affinity, in vivo efficacy, or manufacturing potential.

[0249] Therefore, a series of different amino acid substitution variants were generated using the tool antibody (mAb A). These substitutions were incorporated along with the amino acid substitutions necessary to confer improved serum half-life. A table showing the generated Fc variants is provided (Table 6).

[0250] Table 6. Table of mAb A Fc variants and other Fc variants generated and used in the study. Note that when the molecule name includes LS, it has the M428L / N434S modification to extend the half-life.

[0251]

[0252]

[0253] Protein analysis developability profiles were performed, including i) protein yield, heparin binding, serum stability, nano DSF and HIC to assess the potential for efficient production, ii) human FcRn and CSP target binding to provide confidence that serum half-life and target binding will not be compromised, and iii) human FcγR binding by SPR to evaluate the potential for improved effector function. Complement C1q binding ELISA studies were also performed to evaluate the potential for complement activation by each antibody variant. In addition to in vitro human FcγR binding studies, the selected variants were further evaluated for the potential for improved effector function using an in vitro functional ADNKA assay.

[0254] The yield data from the 2.0 liter scale Fc variant antibody production are shown in Table 7. Compared to the other Fc variants with a total protein yield of greater than 150 mg overall, the Fc variants mAb-A-DEL-LS and mAb A-DE-LS showed surprisingly low total protein yields (30 and 45 mg, respectively, from 2.0 liters of HEK culture). As an additional comparison, the yield of mAb-A produced as a defucosylated molecule from 4 liters of CHO cell culture was very low. Thus, for a given antibody, different amino acid substitutions have significantly different effects on protein yield.

[0255] Table 7 Fc variant antibody production data from 2.0 or 4.0 liters of HEK cell culture and two defucosylated variants from 4 liters of CHO cell culture.

[0256]

[0257] The human FcγR binding data by SPR are shown in Table 8. The affinity data show that two variants (mAb A-VLPLL-LS and mAb A-LPLL-LS) with significantly different affinities between alleles of the same receptor have many different FcγR binding profiles. Between variants, there are obvious differences in binding affinity to FcγR IIIa, with mAb A-AAA-LS being the lowest and mAb A-DEL-LS being the highest. There are also significant differences in FcγR IIa / IIb binding ratios. Between FcγR detection sets, mAb-B-DE-LS shows a very similar binding profile to mAb-A-DE-LS, indicating that a specific set of amino acid substitutions can confer similar improved binding affinity to human FcγR in different antibodies.

[0258] Table 8 shows the SPR affinity data of Fc variants using human FcγR. NB = No Binding, NSB = No Significant Binding

[0259]

[0260]

[0261] Nano-DSF data (Table 9) show a clear melting profile trend, with all variants containing DEA, DEL, and DEAL having Tm1 melting inflection points at or below 50°C, followed by a group of variants with Tm1 in the 51-60°C range, and three variants with Tm1 at 60-65°C. The Tm1 of the mAb-A (wild-type) molecule was 69.45°C. The Tm1 of mAb-B-DE-LS was 51.39°C and was very close to the measured Tm1 of mAb-A-DE-LS (51.13°C), showing that a specific set of amino acid substitutions can have similar effects between different antibodies. Tagg (the onset of aggregation) was fairly consistent between mAb-A variant molecules at 72 / 73°C, which in each case may reflect the melting of the Fab, while the Tagg of mAb-B was several degrees lower. The variable Tm1 melting profiles may indicate reversible melting of the CH2 region.

[0262] Table 9 nano-DSF shows the melting profiles of the Fc variant detection panel.

[0263]

[0264]

[0265] Literature has shown that heparin binding is closely associated with poor antibody pharmacokinetics in vivo (Datta-Mannan et al., 2015 Mabs, 7(3):483-493), and the positive control antibody used in the heparin binding assay is known to have poor pharmacokinetics. Heparin binding ELISA data ( Figure 3 ) showed that all Fc variants exhibited heparin binding lower than the positive control antibody but higher than the negative control. The Fc variants mAb A-DE-LS, mAb A-LS-defucosylated and mAb A-LPL-LS showed the highest mean heparin binding values ​​in this assay.

[0266] Complement C1q ELISA assay data (Table 10) show highly variable binding profiles between different Fc variants. Variants mAb A-IE, mAb A-AE and mAb-A SD have high C1q binding, while variants containing DE (particularly mAb A-DEL-LS and mAb A-DEAL-LS) and mAb A-ALE-LS generally have reduced C1q binding at or near background levels. In addition, different CSP antibodies have different C1q binding profiles, such as mAb A-LS and mAb B-LS.

[0267] Table 10 Complement C1q ELISA binding data on the test panel for Fc variants.

[0268]

[0269]

[0270] Human FcRn binding by SPR (Table 11) showed that the introduction of a series of different amino acid substitutions to improve effector function did not alter binding to human FcRn and therefore would not be expected to significantly affect in vivo pharmacokinetics by themselves.

[0271] Table 11 Fc enhancing amino acid substitutions do not alter human FcRn binding affinity (NB = non-binding, WB = weak binding).

[0272]

[0273]

[0274] The target CSP binding affinity was not altered by the introduction of the Fc substitutions (Table 12), and similarly, hydrophobic interaction chromatography (HIC) did not reveal any significant differences between the Fc variants (Table 13).

[0275] The in vitro stability of the anti-CSP monoclonal antibodies of a test set after incubation in human serum was determined over an 8-week period. Each antibody was spiked into a pooled stock solution of human serum (n=12 donors) to a concentration of 120 μg / ml. Seven individual samples were prepared from each stock solution, and in each case, except for one sample (designated as T0), all samples were incubated at 37°C for a predetermined period (1, 2, 3, 4, 5 or 8 weeks). The T0 sample was immediately frozen on dry ice and then stored at -80°C until all subsequent samples had been collected and frozen in a similar manner.

[0276] Using CSP coated plates for capture and sulfo-tagged anti-human IgG1 Fc mAb for detection, an MSD (Meso Scale Discovery) immunoassay was used to quantify the functional antibodies remaining in each set of samples after incubation. A standard curve was prepared (using unstressed sample matching materials), and the test samples were interpolated and the concentrations were determined based on the standard curve. The recovery from each sample was then expressed as a percentage of its initial T0 sample.

[0277] Although human serum stability data showed no differences between Fc variants of the same molecule ( Figure 4 ), but there are detectable differences between different antibodies (e.g., mAb A molecules and mAb B molecules).

[0278] Table 12: Amino acid substitutions that enhance Fc do not alter CSP target binding affinity

[0279] Antibody Ka(1 / Ms) Kd(1 / s) KD(M) mAb A-SD-LS 1.11E+06 2.02E-04 1.82E-10 mAb A-IE-LS 2.34E+06 7.02E-04 3.01E-10 mAb A-DE-LS 1.77E+06 4.85E-04 2.73E-10 mAb A-DEL-LS 2.80E+06 7.88E-04 2.81E-10 mAb A-AE-LS 1.78E+06 5.03E-04 2.82E-10 mAb A-DEA-LS 1.82E+06 6.80E-04 3.73E-10 mAb A-DEAL-LS 1.49E+06 3.53E-04 2.36E-10 mAb A-FTDE-LS 2.00E+06 4.05E-04 2.03E-10 mAb A-FTAE-LS 1.11E+06 2.21E-04 2.00E-10 mAb A-AAA-LS 1.38E+06 2.50E-04 1.81E-10 mAb A-LPLL-LS 1.99E+06 4.15E-04 2.09E-10 mAb A-LPLIL-LS 2.37E+06 5.63E-04 2.38E-10 mAb A-VLPLL-LS 1.47E+06 3.18E-04 2.16E-10 mAb A-LPL-LS 1.73E+06 5.67E-04 3.28E-10 mAb A-ALE-LS 1.75E+06 3.56E-04 2.03E-10 mAb A-LS 1.22E+06 2.52E-04 2.07E-10 mAb A-defucosylated 1.93E+06 1.96E-04 1.02E-10 mAb A-defucosylated-LS 2.14E+06 3.88E-04 1.81E-10

[0280] Table 13: Fc-enhancing amino acid substitutions do not change HIC retention time

[0281] name RT Peak area Main area% 50% peak width hIgG1 control (replicate 1) 1.239 1965.01 100 0.075 hIgG1 control (replicate 2) 1.251 1893.93 100 0.074 mAb A-SD-LS 1.722 2290.74 100 0.063 mAb A-IE-LS 1.718 2088.56 100 0.063 mAb A-DE-LS 1.724 2003.21 93.33 0.063 mAb A-DEL-LS 1.73 2319 94.82 0.061 mAb A-AE-LS 1.728 2197.18 100 0.063 mAb A-DEA-LS 1.736 1895 100 0.063 mAb A-DEAL-LS 1.741 2274.36 100 0.062 mAb A-FTDE-LS 1.734 2227.51 100 0.062 mAb A-FTAE-LS 1.737 2499.15 100 0.062 mAb A-AAA-LS 1.731 2435.51 100 0.062 mAb A-LPLL-LS 1.744 2379.66 100 0.063 mAb A-LPLIL-LS 1.744 2138.34 100 0.064 mAb A-VLPLL-LS 1.746 2093.79 100 0.062 mAb A-LPL-LS 1.73 2381.57 100 0.062 mAb A-ALE-LS 1.735 2492.77 100 0.061 mAb A-LS (Batch 1) 1.716 2963.19 100 0.065 mAb A-LS (Batch 2) 1.718 2710.42 100 0.06 mAb A 1.687 2866.02 100 0.059 mAb A-Fc silencing 1.694 2593.69 100 0.058 mAb A-DE-LS 1.725 2724.26 100 0.058 mAb B 1.17 2701.41 100 0.065 mAb B-LS 1.218 2711.34 100 0.064 mAb B-DE-LS 1.216 2758.31 100 0.064 mAb A-defucosylated 1.693 2424.78 100 0.064 mAb A-defucosylated-LS 1.723 2493.09 100 0.064

[0282] Nine Fc variants were selected based on the above data, which have good physical properties and predicted enhanced Fc functions (based on FcγR binding affinity). The data generated by in vitro human antibody-dependent NK cell activation (ADNKA) assay (Table 14) (wherein recombinant CSP is coated on a microtiter plate, incubated with antibodies, and then incubated with NK cells from human donors) were used to further classify these Fc variants into three types. NK activation markers were then measured by flow cytometry. These studies show that all tested Fc modifications enhance ADNKA in the presence of recombinant CSP (particularly those recombinant CSPs containing "DE" mutations and "LPLIL" variants). DEA, FTDE and ALE were selected from the detection set to ensure that based on the published examples and by the binding affinity with human FcγR shown in Table 8, the variants advanced for further evaluation represent those variants that will enhance the full range of Fc-dependent mechanisms. As a result of this work, and to address the low Tm1 temperature associated with the DEA variant, the T250V / A287F (V / F) mutations were added to the DEA variant for future studies.

[0283] Table 14 In vitro evaluation of Fc-enhanced antibodies in the antibody-dependent natural killer cell activation (ADNKA) assay

[0284]

[0285]

[0286] Example 4 Incorporating amino acid substitutions that confer optimal Fc effector function into the most potent lead antibodies

[0287] The amino acid substitutions selected from Example 3 were each incorporated into the sequences of the most potent antibodies mAb 356 and mAb 338 identified in the in vivo study of Example 2. Functional in vitro assays were performed to confirm that the improved effector function can be transferred to these most potent molecules identified by in vivo studies. The molecules generated were 338-ALE-LS, 356-ALE-LS and 356-DEA-FV-LS. The FTDE-LS variant was not further evaluated. In the ADCP assay measuring the phagocytosis of the beads coated with recombinant CSP by the monocytic cell line THP-1, all tested molecules were more potent than mAb A comparators (including mAb A variants modified with DE and FTAE Fc). Two 356 variants 356-ALE-LS and 356-DEA-FV-LS were more potent than any other tested molecules of EC50<1pM (Table 15). In the ADNKA assay, all ALE- and DEA-FV-containing molecules elicited enhanced ADNKA compared to their LS-only versions (Table 16).

[0288] Table 15a In vitro evaluation of lead antibodies in antibody-dependent cellular phagocytosis (ADCP) assay - average from 2 replicates

[0289]

[0290] Table 15b In vitro evaluation of lead antibodies in replicates in the (ADCP) assay, with 4 replicates per average

[0291]

[0292] Table 16 Geometric mean EC50 (ug / ml) of lead antibodies in vitro evaluation in antibody-dependent natural killer cell activation assay (ADNKA)

[0293] mAbs Donor 1 Donor 2 Donor 3 Donor 4 317LS 0.0567 0.231 0.1513 0.0709 CIS43LS 0.0277 0.1426 0.1044 0.0643 L9 LS 0.038 0.5143 0.0887 0.0286 317DE-LS 0.0027 0.0022 0.0276 0.0111 AB-000356ALE LS 0.0052 0.0035 0.1886 0.0104 AB-000338LS 0.0301 0.2376 0.1714 0.0887 AB-000356LS 0.0704 1.5013 1.3069 0.1094 AB-000338ALE LS 0.0035 0.0037 0.0802 0.0214 AB-000356DEA FV LS 0.0038 0.0019 0.0165 0.0054 AB-000338DEA FV LS 0.0087 0.0057 0.0272 0.0067

[0294] To evaluate the in vivo impact of improved effector function, antibodies 356-ALE-LS and 356-DEA-FV-LS were compared with mAb 356-LS in a P. berghei mouse model. The study protocol is shown in Table 17 and the data are shown in Figure 5 Also shown are the serum concentrations of each mAb at 2, 24, 44, and 96 hours after dosing. EpoFix (100 μg dose) and mAbA-LS (25 μg and 100 μg doses) were used as positive and negative controls for infection.

[0295] Table 17

[0296]

[0297] Statistical analysis of the PK / PD relationship between 356-ALE-LS and 356-LS showed a small effect of Fc enhancement, with the probability of the Fc-enhanced mAb 356-ALE-LS being at least 1-fold more potent than 356-LS approaching or exceeding 90%. The specific probabilities were 87.60% on day 2, 99.96% on day 3, and 94.96% on day 5. For a three-fold improvement in protective efficacy compared to the 356-LS molecule, these probabilities dropped to 21.06% (day 2), 61.76% (day 3), and 5.55% (day 5).

[0298] This very small difference in potency between 356-ALE-LS and 356-LS observed in the P. berghei model is inconsistent with the larger difference observed in the in vitro functional Fc assay described in Example 4. Given the known differences between human and mouse FcγR activity and cellular distribution, it is possible that the mouse C57BL / 6 mouse background does not reflect levels of Fc-enhanced antibody activity consistent with human mAbs, and therefore further in vivo studies will use mice transgenic for human FcγRs.

[0299] Example 5—Safety Analysis of Lead Antibodies

[0300] The four lead antibodies were subjected to various assays and tests to set up the (in vitro) background as closely as possible to ensure that the development of these antibodies will be safe in terms of e.g. immunogenicity, tissue cross-reactivity, etc. A brief summary of the results is as follows:

[0301] i) Human peripheral blood mononuclear cells from 20 different donors were used as a source of CD4+T cells and dendritic cells, and CD4+T cell proliferation and IFNγ production were used as readouts to test the immunogenicity of the four leads 356-ALE-LS, 356-DEA-FV-LS, 338-LS and 338-ALE-LS. The results provide an overall assessment between the four molecules, with 356-ALE-LS inducing 2 positive reactions in 17 donors (12% reaction rate), 356-DEA-FV-LS inducing 5 positive reactions in 17 donors (29% reaction rate), and 338-ALE-LS and 338-LS both inducing 3 positive reactions in 16 donors (19% reaction rate). Based on the assay certification results and the cumulative data from the internal immunogenicity risk testing, these results indicate that these mAbs may exhibit similar potential to induce immune responses.

[0302] ii) Non-GLP human tissue cross-reactivity (TCR) was performed with the four lead mAbs in testis, kidney, skin, tonsil, and eye tissues, and no positive staining for non-specific binding to the mAbs was shown in any of the five human tissues.

[0303] iii) Human off-target binding assessment was performed using cell microarray technology (Retrogenix), in which a detection set of full-length plasma membrane proteins, plasma membrane tethered-secreted proteins and a set of heterodimers (more than 6400 human proteins) were tested for binding to 4 mAb leads at 20 μg / ml mAb. Specific interactions were observed between the SPOCK1 protein and two of these mAbs (356-ALE-LS and 356-DEA-FV-LS). mAbs 338-LS and 338-ALE-LS showed specific interactions with CNTNAP2. To further investigate these results, further SPR binding data were generated using recombinant SPOCK1 and CNTNAP2 proteins and these 4 mAbs. These SPR results confirmed that none of the antibodies showed non-specific binding to the surface with binding higher than 300 nM protein.

[0304] iv) SCISSOR (Subcutaneous Injection Site Simulator) is an in vitro assay designed to test the bioavailability of molecules after subcutaneous injection. The clinical bioavailability prediction value of GSK4425689A from SCISSOR analysis is 51.90% (NCR2022N506638_00). As background information, this value is similar to the bioavailability of a small group of approved comparator mAbs between 52 and 80% (Zhao L et al. 2013).

[0305] Example 6 In vitro and in vivo evaluation of Fc-enhancing mAbs in mice transgenic for the human Fcγ receptor

[0306] Although there are examples of Fc-enhanced mAbs with improved efficacy using mouse models (Hiatt et al., 2014, PNAS 111; 5992-5997; DiLillo et al., 2014, Nat Med 20 (2) 143-151), the known differences in effector cell distribution between human and mouse Fcγ receptors make the translational value of wild-type mouse models uncertain. Therefore, mice transgenic for human Fcγ receptors have been previously developed (Smith et al., 2012, Proc Natl Acad Sci US A., 109 (16): 6181-6186) and used to better predict the translational value of mAbs in diseases (Casey et al., 2018, Leukemia, 32 (2): 547-549).

[0307] Our previous studies have shown a large discrepancy between >20-fold improvement in in vitro activity of Fc-enhanced molecules compared to very small in vivo improvements in C57BL / 6 mice, and therefore we will explore the possibility that the C57BL / 6 mouse P. berghei model may not represent the translational efficacy of Fc-enhanced mAbs using mice transgenic for the human Fcγ receptor.

[0308] In vitro studies aimed at validating the transgenic mice will be performed, specifically aimed at showing that NK cells isolated from transgenic mice (but not from C57BL / 6 mice) respond to activation in an assay using antibodies bound to immobilized CSP. These data will provide confidence that transgenic mice better reflect the true translational value of mAbs designed to enhance NK cells (and other effector cells) against malaria sporozoites and further explain why C57BL / 6 mice respond poorly to such Fc-enhanced mAbs.

[0309] Following the in vitro work, in vivo studies were performed using these transgenic mice to demonstrate agreement with our previous studies in C57BL / 6 mice infected with P. berghei (shown in Figure 5 Compared with the Fc-enhanced mAbs, the Fc-enhanced mAbs showed a wider range of potency.

[0310] Example 7 In vitro and in vivo evaluation of Fc-enhancing mAbs in mice transgenic for the human Fcγ receptor.

[0311] In vitro studies to evaluate the activation potential of NK cells isolated from both wild-type C57BL / 6 mice and mice transgenic for human Fcγ receptors involved coating recombinant CSP onto microtiter plates and incubating with anti-CSP mAb followed by the addition of NK isolated from mouse splenocytes. The activation marker CD107a was measured by flow cytometry. The difference in response between mAb AB-000356LS and AB-000356ALE LS in human FcγR transgenic mice showed that enhanced Fc replacement can enhance NK cell activation ( Figure 6 a), while studies using wild-type C57 / Bl6 mice ( Figure 6 b) shows no NK activation signal above baseline. These in vitro studies may partially explain why C57BL / 6 mice respond poorly to Fc-enhanced mAbs.

[0312] Then, in vivo studies were performed in mice transgenic for human Fcγ receptors. Briefly, two antibody doses (25 μg and 100 μg per mouse) were administered by iv route to transgenic mice (n=5 per group) and WT C57BL / 6 mice (n=4 per group) 24 hours before challenge with 1500 sporozoites. Liver infection was evaluated at 44 hours, and blood stage infection was assessed by PCR on day 3 and by FACS on days 5, 6, and 8. The antibody variants used in this study were 356-LS, 356-LAGA, and 356ALE-LS. A negative control antibody (EpoFix) was also included in this study. The blood parasitemia levels on day 3 after sporozoite challenge are shown in Figure 7 The data are summarized in Tables 18 and 19. There was no significant difference in the level of protection between the different antibodies, and all antibodies showed similar levels of protection compared to the negative control antibody. The pharmacokinetic data showed that the antibody concentrations in the blood were roughly similar between the molecules.

[0313] Table 18 Study data summary: 25 μg dose group

[0314]

[0315]

[0316] Table 19 Summary of study data: 100 μg dose group

[0317]

[0318]

[0319] Although NK cells isolated from human transgenic mice were shown to respond robustly to Fc-enhanced 356-ALE-LS, no significant increase in protection was observed following sporozoite challenge. The reasons for this are unclear but may be due to a range of factors, including differences between the mouse challenge model and humans, for example, the presence and activity of specific effector cell subsets thought to be involved in protection against human malaria disease (Ty et al., 2023, Sci Transl Med, 15(680):eadd901), which have not been evaluated in mice; the difference in the time interval between infectious sporozoite challenge and the end of the liver stage of disease in mice compared with humans (approximately 2 days versus 7 days, respectively), and the regimen used in these iv challenge studies does not reflect the natural mosquito bite route of infection.

[0320] Example 8 - In vivo evaluation of antibodies in mice implanted with human hepatocytes and infected with Plasmodium falciparum

[0321] To evaluate the in vivo effectiveness of 356-ALE-LS against human malaria Plasmodium falciparum, a human liver chimeric FRGhuHep mouse model was used (Minkah et al., 2018, Front Immunol, 19(9):807). These mice were implanted with primary human hepatocytes, susceptible to infection with Plasmodium falciparum sporozoites, and supported complete liver stage development. Mice (n=6 per group) were challenged by mosquito bites using 40 infected mosquitoes, and 356-ALE-LS (Group 2) was administered at a single 300 μg dose per mouse. Antibody L9-LS (Group 3) has been shown to be very effective in the FRG model, and human malaria challenge studies have recently been completed in humans, which was also included in the study as a positive baseline control. EpoFix isotype antibody was used as a negative control (Group 1). All six mice treated with 356-ALE-LS had undetectable parasitemia in peripheral blood on day 8 after sporozoite challenge, which was similar to the fully protected 5 of 6 mice in the L9-LS group ( Figure 8 C). No differences in antibody serum concentrations were observed across the study ( Figure 8 D) The data show that 356-ALE-LS is comparable to L9-LS, although the additional benefit of improved effector function would not be apparent due to the WTC57BL / 6 background strain of mice used.

[0322] The binding affinity of 356-ALE-LS to recombinant CSP was measured by SPR. The data are shown in Table 20. Two different batches of 356ALE-LS were shown to bind to the target recombinant CSP with similar nM affinity.

[0323] Table 20 Binding affinity of antibodies to recombinant CSP by SPR

[0324]

[0325] The binding affinity of 356ALE-LS to human Fcγ receptors was measured by SPR. The binding affinity of 356LS and the Fc silent variant 356LAGA-LS was also determined. Human IgG1 antibodies in WT and Fc silent (LAGA) format were included as negative controls. The data are shown in Table 21. Compared to the parental 356-LS mAb, mAb 356-ALE-LS showed higher human FcγRIIa and FcγRIIIa binding affinity, with similar low hFcγRIIb binding.

[0326] Table 21 Binding affinity of antibodies to human FcγR by SPR

[0327]

[0328]

[0329] WB = Weak Binding

[0330] The binding affinity of 356ALE-LS to human FcRn was measured by SPR. The binding affinity of mAb-LS and two controls (human IgG1 WT antibody and antibody L9 without LS) was also measured. The data are shown in Table 22, where both 356-ALE-LS and mAbA LS showed the expected lower binding affinity at pH 6.0.

[0331] Table 22 Human FcRn binding by SPR

[0332] mAbs KD at pH 6.0 (nM) KD at pH 7.0 (nM) hIgG1 WT control 102 NB 356-ALE-LS 52 WB mAb A-LS 62 WB L9 174 NB

[0333] WB = Weak Binding

[0334] NB = No Binding

[0335] Sequence Listing

[0336] CDRH1 of SEQ ID NO:1:356

[0337]

[0338] SEQ ID NO:2:CDRH2 356

[0339]

[0340] SEQ ID NO:3:CDRH3

[0341]

[0342] SEQ ID NO:4:CDRL1

[0343]

[0344] SEQ ID NO:5:CDRL2

[0345]

[0346] SEQ ID NO:6:CDRL3

[0347]

[0348] SEQ ID NO:7: variable heavy chain

[0349]

[0350] SEQ ID NO:8: variable light chain

[0351]

[0352] SEQ ID NO:9: Heavy chain

[0353]

[0354] SEQ ID NO: 10: Light chain

[0355]

[0356] SEQ ID NO:11CDRH1

[0357]

[0358] SEQ ID NO:12CDRH2

[0359]

[0360] SEQ ID NO:13CDRH3

[0361]

[0362] SEQ ID NO:14CDRL1

[0363]

[0364] SEQ ID NO:15CDRL2

[0365]

[0366] SEQ ID NO:16CDRL3

[0367]

[0368] SEQ ID NO: 17 variable heavy chain

[0369]

[0370] SEQ ID NO: 18 variable light chain

[0371]

[0372] SEQ ID NO: 19 Heavy chain

[0373]

[0374] SEQ ID NO:20 Light chain

[0375]

[0376] SEQ ID NO:21 Variable heavy chain nucleotide sequence (356)

[0377]

[0378] SEQ ID NO:22 Variable light chain nucleotide sequence (356)

[0379]

[0380] SEQ ID NO:23 Heavy chain nucleotide sequence

[0381]

[0382] SEQ ID NO:24 Variable light chain nucleotide sequence (356)

[0383]

[0384] SEQ ID NO:25 Variable heavy chain nucleotide sequence (338)

[0385]

[0386] SEQ ID NO:26 Variable light chain nucleotide sequence (338)

[0387]

[0388] SEQ ID NO:27 Heavy chain nucleotide sequence (338ALE LS)

[0389]

[0390] SEQ ID NO:28 Light chain nucleotide sequence (338ALE LS)

[0391]

[0392] SEQ ID NO:29 356DEA FV LS heavy chain

[0393]

[0394] SEQ ID NO:30 356DEA FV LS light chain

[0395]

[0396] SEQ ID NO:31 356 heavy chain nucleotide sequence

[0397]

[0398] SEQ ID NO:32 356DEA FV LS heavy chain nucleotide sequence

[0399]

Claims

1. A Plasmodium falciparum circumsporozoite protein (CSP) antigen binding protein comprising: The heavy chain (HC) sequence of SEQ ID NO:9; and The light chain (LC) sequence of SEQ ID NO:

10.

2. The CSP antigen binding protein of claim 1, wherein the antigen binding protein binds with an affinity of at least 2.5 nM when measured by surface plasmon resonance (SPR).

3. The CSP binding protein of claim 1 or claim 2, wherein the antigen binding protein remains in the circulation for a period of at least 3 months after administration.

4. A nucleic acid sequence encoding the heavy chain of SEQ ID NO: 9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO:

10.

5. One or more expression vectors comprising a nucleic acid sequence encoding the heavy chain of SEQ ID NO: 9; and / or a nucleic acid sequence encoding the light chain of SEQ ID NO:

10.

6. A recombinant host cell comprising one or more nucleic acid sequences as defined in claim 4 or one or more expression vectors as defined in claim 5.

7. A method for producing the CSP antigen binding protein according to claims 1 to 3, said method comprising culturing a host cell as defined in claim 6 under conditions suitable for expressing said one or more nucleic acid sequences or one or more vectors, thereby producing a polypeptide comprising said CSP antigen binding protein.

8. A pharmaceutical composition comprising a CSP antigen binding protein as defined in any one of claims 1 to 3 and a pharmaceutically acceptable excipient.

9. A method for preventing malaria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CSP antigen binding protein as defined in any one of claims 1 to 3 or a pharmaceutical composition as defined in claim 8.

10. A CSP antigen binding protein as defined in any one of claims 1 to 3 or a pharmaceutical composition as defined in claim 8 for use in preventing malaria.

11. A CSP antigen binding protein as defined in any one of claims 1 to 3 or a pharmaceutical composition as defined in claim 8 for intravenous, subcutaneous or intramuscular administration.

12. Use of a CSP binding protein as defined in any one of claims 1 to 3 or a pharmaceutical composition as defined in claim 8 for the preparation of a medicament for preventing malaria.

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