Antigen-binding proteins and uses thereof

Abstract

The present invention provides antigen-binding proteins, such as antibodies, that target Plasmodium sporozoites, more particularly Plasmodium circumsporozoite proteins. The present invention also provides nucleic acids encoding such antibodies. The present invention further provides antibodies according to the present invention for use in the prevention of malaria.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the field of malaria treatments, in particular to antibodies for the prevention of malaria that bind to Plasmodium falciparum sporozoites, in particular to Plasmodium falciparum circumsporozoite proteins. [Background technology]

[0002] Background of the Invention Malaria is one of the most serious public health problems worldwide. It is caused by parasitic protozoa of the genus Plasmodium. The genus Plasmodium includes approximately 200 species, but four species—P. falciparum, P. vivax, P. ovale, and P. malariae—account for nearly all human infections caused by Plasmodium. Among these Plasmodium species, P. falciparum is responsible for the vast majority of malaria deaths, especially in children under the age of five. Symptoms of malaria typically include fever, fatigue, vomiting, and headache. Severe cases can result in yellow skin, convulsions, coma, or death.

[0003] Malaria is a mosquito-borne disease transmitted by the bite of infected female Anopheles mosquitoes. In human malaria caused by Plasmodium falciparum infection, female Anopheles mosquitoes inject a small number of sporozoites (10–100) into the skin. Some of these parasites migrate to the liver and invade hepatocytes (Crompton et al. (2014) Annu Rev Immunol 32, 157–187). Within hepatocytes, sporozoites form parasitophorous vacuoles, where they develop and multiply asexually (schizogony), maturing into schizonts that rupture and release thousands of merozoites, which are eventually released into the bloodstream. Merozoites infect red blood cells and mature into rings, then trophozoites, and finally schizonts, which rupture and release merozoites in a 48-hour cycle that infect new red blood cells, perpetuating the cycle. Other merozoites undergo sexual differentiation into the erythrocytic stage (gametocytes). When a mosquito bites an infected human, gametocytes are ingested with the blood and mature in the mosquito's gut. Male and female gametocytes combine to form ookinetes, fertilized, motile zygotes. The ookinetes further develop into oocysts and finally new sporozoites, which migrate to the insect's salivary glands and infect new vertebrate hosts.

[0004] Malaria symptoms are caused by blood-stage parasites. However, sporozoites, in contrast, do not cause clinical symptoms, and the sporozoite and liver stages of the Plasmodium parasite life cycle are present in small numbers in the host, so eradicating them can completely eliminate the infection. Therefore, the sporozoite and liver stages of the Plasmodium falciparum parasite are important targets for current malaria prophylactic drug candidates, because successful interventions to protect against these stages can prevent both infection and transmission of malaria. Therefore, vaccines or molecules that block sporozoites are central to the development of malaria prophylactic drugs.

[0005] The Plasmodium circumsporozoite protein (CSP) is a membrane-bound protein present only in the sporozoite stage of Plasmodium parasites. It has been hypothesized that CSP forms a dense capsule on the parasite surface and mediates many of the initial interactions between sporozoites and 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 among various Plasmodium species that infect humans, nonhuman primates, and rodents. The amino acid sequence of CSP contains an immunodominant central repeat region (NANP repeat region in the case of Plasmodium falciparum) that differs among Plasmodium species. Adjacent to this repeat are two conserved motifs, including a known cell adhesion motif at the C-terminus of the repeat, called type I thrombospondin repeats (TSRs). These conserved motifs are involved in protein processing during parasite transfer from mosquitoes to mammalian vectors. CSPs are known to play an important role in the movement of sporozoites from the midgut wall of infected mosquitoes to the mosquito's salivary glands. Furthermore, CSPs are also involved in hepatocyte binding in mammalian hosts, where the N-terminus of CSPs initially promotes parasite binding. On the hepatocyte surface, proteolytic cleavage at the N-terminal I region exposes the adhesive C-terminal domain, thereby directing the parasite toward liver invasion (Coppi et al., 2005, J Exp Med 201, 27-33).

[0006] The current leading malaria vaccine candidate is RTS,S (RTS,S / AS01; trade name Mosquirix), a recombinant protein-based malaria vaccine. RTS,S is a hybrid protein particle formulated in a multicomponent adjuvant called AS01. The RTS,S vaccine antigen consists of 19 NANP amino acid repeat units followed by the complete C-terminal domain of the GPI-anchored CSP antigen fused to the S protein of hepatitis B virus. A multisite clinical trial in sub-Saharan Africa has shown that RTS,S provides moderate, 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 prophylaxis is the difficulty of characterizing the mechanisms that confer robust immune protection, although recent progress has been made in identifying immune correlates related to antibody specificity and function (Suscovich et al, 2020, Sci Transl Med 12(553):eabb4757).

[0008] Recently, antimalarial antibodies specific for Plasmodium falciparum CSPs have been reported (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 reported to be protective in preclinical mouse infection models, their mechanism of action has been suggested to be solely via the antigen-binding Fab region of the antibodies (e.g., CIS43, L9) to immobilize sporozoites in the skin or to prevent sporozoites from binding to and / or infecting target cells in the liver. There is growing recognition that antibody Fc-mediated effector mechanisms may play an important role in infection protection and that manipulating these mechanisms using amino acid substitutions in the Fc region of antibodies can significantly improve the overall level of protection conferred by antibodies. A recently published immune correlation study (Suscovich et al., 2020, Sci Transl Med 12(553):eabb4757) points to such a role in malaria, but other studies have previously demonstrated this principle in other infectious disease conditions (Hiatt et al., 2014, PNAS 111; 5992-5997; DiLillo et al., 2014, Nat Med 20(2) 143-151). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Crompton et al. (2014) Annu Rev lmmunol 32, 157-187) [Non-patent document 2] Menard, 2000, Microbes Infect. 2:633-642; [Non-patent document 3] Sinnis and Nardin, 2002, Chem Immunol 80:70-96 [Non-Patent Document 4] Coppi et al, 2005, J Exp Med 201, 27-33

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Summary of the Invention

Problems to be Solved by the Invention

[0010] Therefore, it is an object of the present invention to overcome the limitations of the prior art malaria treatments described above by providing antibodies that not only have high affinity, but also offer the advantages of ease of development as well as other protective mechanisms and improved safety, taking into account the inherent problems of administration in malaria-endemic countries. [Means for solving the problem]

[0011] Summary of the Invention In one aspect of the invention, there is provided an antigen binding protein of Plasmodium falciparum.

[0012] In one embodiment, The heavy chain (HC) sequence of SEQ ID NO: 9; and Light chain (LC) sequence of SEQ ID NO: 10 The present invention provides a Plasmodium falciparum circumsporozoite protein (CSP) antigen binding protein comprising:

[0013] In certain embodiments, 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 is provided.

[0014] In certain embodiments, one or more expression vectors are provided that comprise 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 certain embodiments, a recombinant host cell is provided that comprises one or more nucleic acid sequences 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 that comprise 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 there is provided a method for producing a circumsporozoite protein (CSP) antigen binding protein of the invention disclosed herein, which method comprises culturing a host cell under conditions suitable for expression of one or more of said nucleic acid sequences or one or more vectors of the invention described herein, whereby a polypeptide comprising the CSP antigen binding protein is produced.

[0017] In certain embodiments, a pharmaceutical composition is provided comprising a CSP antigen binding protein of the invention described herein and a pharmaceutically acceptable excipient.

[0018] In one embodiment, a method is provided for preventing malaria in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CSP antigen binding protein of the invention as described herein, or a pharmaceutical composition comprising a CSP antigen binding protein of the invention as described herein.

[0019] In one embodiment, the CSP antigen binding proteins of the invention described herein, or pharmaceutical compositions comprising the CSP antigen binding proteins of the invention described herein, are for use in the prevention of malaria. [Brief explanation of the drawings]

[0020] [Figure 1] Diversity analysis of hit antibody panel [Figure 2] Heparin-binding ELISA data for the hit antibody panel, including AB-000325, AB-000337, AB001446, and AB-001516, show various molecules with higher heparin-binding properties than the negative control, which were subsequently excluded from further analysis. Antibodies were incubated on heparin-coated poly-L-lysine plates for 2 hours before detection with anti-F(ab)'2-HRP. [Figure 3]Heparin binding ELISA data for a panel of antibody Fc variants. Heparin binding ELISA data for a panel of mAb A antibodies harboring various amino acid substitutions to confer extended half-life and improved effector function. [Figure 4] Human serum stability of a panel of antibody Fc variants. mAb-A Fc variants show no significant differences in stability in human plasma, but different molecules (i.e., mAb-A vs. mAb-B) have inherently different stabilities. In vitro human serum stability over 8 weeks is demonstrated using CSP-coated plates for capture and a sulfo-tagged anti-human IgG1 Fc mAb for antibody detection. [Figure 5a] In vivo evaluation of the effect of Fc enhancement on antibody efficacy in a P. berghei model. Figure 5a shows liver burden measured by bioluminescence at 44 hours. Figure 5b shows day 5 (percent infected red blood cells) after sporozoite challenge at a 25 μg dose. Figure 5c shows day 8 (percent infected red blood cells) after sporozoite challenge at a 100 mg dose. Figure 5d shows pharmacokinetic blood concentrations of antibodies at 2, 24, 44, and 96 hours after sporozoite challenge. Statistical analysis of 356-ALE-LS with 356-LS, taking into account differences in serum concentrations between the respective mAbs, revealed that the effect of Fc enhancement was very small, with the probability of 356-ALE-LS being at least 1-fold more potent than 356-LS being nearly 90% or greater (87.60% on day 2, 99.97% on day 3, and 94.96% on day 5). This probability decreased to 21.06% (day 2), 63.05% (day 3), and 5.55% (day 5) for a three-fold change. [Figure 5b]In vivo evaluation of the effect of Fc enhancement on antibody efficacy in a P. berghei model. Figure 5a shows liver burden measured by bioluminescence at 44 hours. Figure 5b shows day 5 (percent infected red blood cells) after sporozoite challenge at a 25 μg dose. Figure 5c shows day 8 (percent infected red blood cells) after sporozoite challenge at a 100 mg dose. Figure 5d shows pharmacokinetic blood concentrations of antibodies at 2, 24, 44, and 96 hours after sporozoite challenge. Statistical analysis of 356-ALE-LS with 356-LS, taking into account differences in serum concentrations between the respective mAbs, revealed that the effect of Fc enhancement was very small, with the probability of 356-ALE-LS being at least 1-fold more potent than 356-LS being nearly 90% or greater (87.60% on day 2, 99.97% on day 3, and 94.96% on day 5). This probability decreased to 21.06% (day 2), 63.05% (day 3), and 5.55% (day 5) for a three-fold change. [Figure 5c] In vivo evaluation of the effect of Fc enhancement on antibody efficacy in a P. berghei model. Figure 5a shows liver burden measured by bioluminescence at 44 hours. Figure 5b shows day 5 (percent infected red blood cells) after sporozoite challenge at a 25 μg dose. Figure 5c shows day 8 (percent infected red blood cells) after sporozoite challenge at a 100 mg dose. Figure 5d shows pharmacokinetic blood concentrations of antibodies at 2, 24, 44, and 96 hours after sporozoite challenge. Statistical analysis of 356-ALE-LS with 356-LS, taking into account differences in serum concentrations between the respective mAbs, revealed that the effect of Fc enhancement was very small, with the probability of 356-ALE-LS being at least 1-fold more potent than 356-LS being nearly 90% or greater (87.60% on day 2, 99.97% on day 3, and 94.96% on day 5). This probability decreased to 21.06% (day 2), 63.05% (day 3), and 5.55% (day 5) for a three-fold change. [Figure 5d]In vivo evaluation of the effect of Fc enhancement on antibody efficacy in a P. berghei model. Figure 5a shows liver burden measured by bioluminescence at 44 hours. Figure 5b shows day 5 (percent infected red blood cells) after sporozoite challenge at a 25 μg dose. Figure 5c shows day 8 (percent infected red blood cells) after sporozoite challenge at a 100 mg dose. Figure 5d shows pharmacokinetic blood concentrations of antibodies at 2, 24, 44, and 96 hours after sporozoite challenge. Statistical analysis of 356-ALE-LS with 356-LS, taking into account differences in serum concentrations between the respective mAbs, revealed that the effect of Fc enhancement was very small, with the probability of 356-ALE-LS being at least 1-fold more potent than 356-LS being nearly 90% or greater (87.60% on day 2, 99.97% on day 3, and 94.96% on day 5). This probability decreased to 21.06% (day 2), 63.05% (day 3), and 5.55% (day 5) for a three-fold change. [Figure 6] In vitro evaluation of CSP-mediated activation of primary NK cells isolated from wild-type C57BL / 6 mice and human Fcγ receptor transgenic mice. Recombinant CSP protein was coated onto microtiter plates, incubated with anti-CSP antibody, and then NK cells isolated from the spleens of wild-type C57BL / 6 mice or transgenic mice expressing human Fcγ receptors were added and incubated for 3 hours. NK activation was assessed by CD107a staining. [Figure 7] Parasite quantification in peripheral blood on day 3 after sporozoite challenge was measured by real-time PCR. Wild-type C57BL / 6 mice and human FcγR mice were passively transferred (100 μg (left) and 25 μg (right)) with the indicated mAbs (EpoFix as isotype control, 356-LS, 356-ALE-LS, 356-LAGA-LS) 24 h before challenge with 1500 sporozoites. [Figure 8]In vivo neutralizing activity of 356-ALE-LS and L9-LS against Plasmodium falciparum in FRG-huHep. (A) Representative image of parasite liver burden measured by bioluminescence of luciferase-expressing transgenic P. falciparum parasites. (B) Reduction of liver burden (total luminous flux, photons / second) at day 6 post-infection. (C) Protection data at day 8 post-sporozoite challenge. (D) Serum antibody concentration (μg / mL) at the time of parasite challenge.

[0021] Detailed Description of the Invention In one aspect of the invention, a Plasmodium falciparum circumsporozoite protein (CSP) antigen binding protein is provided.

[0022] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5; and a CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein has been modified to have improved effector function and extended half-life.

[0023] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 11, a CDRH2 of SEQ ID NO: 12, a CDRH3 of SEQ ID NO: 13, a CDRL1 of SEQ ID NO: 14, a CDRL2 of SEQ ID NO: 15; and a CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein has been modified to have an improved effector function or an extended half-life.

[0024] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 11, a CDRH2 of SEQ ID NO: 12, a CDRH3 of SEQ ID NO: 13, a CDRL1 of SEQ ID NO: 14, a CDRL2 of SEQ ID NO: 15; and a CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein has been modified to have improved effector function and extended half-life.

[0025] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5; and a CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein has been modified to have an improved effector function or an extended half-life.

[0026] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 11, a CDRH2 of SEQ ID NO: 12, a CDRH3 of SEQ ID NO: 13, a CDRL1 of SEQ ID NO: 14, a CDRL2 of SEQ ID NO: 15; and a CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein has been modified to have an improved effector function or an extended half-life.

[0027] In another embodiment, a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5; and a CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein is mutated at one of the following positions or combinations of positions: i) S239D ii) I332E iii) S239D / I332E iv) S239D / I332E / A330L v) G236A / I332E vi) S239D / I332E / G236A vii) G236A / S239D / I332E / A330L viii) H268F / S324T / G236A / I332E ix) S267E / H268F / S324T / G236A / I332E x) S298A / E333A / K334A xi) F243L / R292P / Y300L / P396L xii) F243L / R292P / Y300L / V305I / P396L xiii) L235V / F243L / R292P / Y300L / P396L xiv) F243L / R292P / Y300L xv) G236A / A330L / I332E xvi) K228M / K334E xvii) S239D / I332E / G236A / T250V / A287F xviii) H268F / S324T / S239D / I332E xix) T250V / A287F / G236A / A330L / I332E xx) T250V / A287F Furthermore, the circumsporozoite protein (CSP) antigen-binding proteins are provided, wherein the effector function of the antigen-binding protein is improved.

[0028] In another embodiment, a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 11, a CDRH2 of SEQ ID NO: 12, a CDRH3 of SEQ ID NO: 13, a CDRL1 of SEQ ID NO: 14, a CDRL2 of SEQ ID NO: 15; and a CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein is mutated at at least one of the following positions or combinations of positions: i) S239D ii) I332E iii) S239D / I332E iv) S239D / I332E / A330L v) G236A / I332E vi) S239D / I332E / G236A vii) G236A / S239D / I332E / A330L viii) H268F / S324T / G236A / I332E ix) S267E / H268F / S324T / G236A / I332E x) S298A / E333A / K334A xi) F243L / R292P / Y300L / P396L xii) F243L / R292P / Y300L / V305I / P396L xiii) L235V / F243L / R292P / Y300L / P396L xiv) F243L / R292P / Y300L xv) G236A / A330L / I332E xvi) K228M / K334E xvii) S239D / I332E / G236A / T250V / A287F xviii) H268F / S324T / S239D / I332E xix) T250V / A287F / G236A / A330L / I332E xx) T250V / A287F Furthermore, the circumsporozoite protein (CSP) antigen-binding proteins are provided, wherein the effector function of the antigen-binding protein is improved.

[0029] Studies in naturally infected human patients have shown that antibody effector functions can develop over time and correlate with improved protection. However, this occurs only after multiple mosquito bites and exposure to P. falciparum malaria parasites, and development can take years (Fenget et al, 2021, Nat Comms 12:1742). Currently, no direct-acting antimalarial antibodies have been shown to have an Fc-mediated role in protection, nor have they been further engineered to improve Fc-mediated host effector cell mechanisms.

[0030] Because of the difficulty in demonstrating cell killing of the parasite and parasite-infected cells by targeting CSPs (which are not naturally expressed on human cells in the absence of infection), we demonstrated improved antimalarial activity by modifying the Fc region to show increased antibody-dependent NK cell activation (ADNKA) and antibody-dependent cellular phagocytosis (ADCP) effector activity, which serve as markers of improved antibody-mediated effector function or ADCC. Thus, the use of mAbs with such improved effector function could enable more effective prevention, especially in young children, if these protective mechanisms can be provided as early as possible.

[0031] An additional advantage of the antigen-binding proteins of the present invention is the formation of immune complexes (ICs). Upon binding to infectious parasites, the antigen-binding proteins bind to antigen-presenting cells, specifically dendritic cells (DCs), and engage the host's adaptive immune response. Subsequent antigen presentation can result in the priming of T-cell and B-cell immune responses targeting infectious antigens. In this way, a "vaccine-like" response against the infectious agent can be elicited, resulting in improved, longer-term immune protection. This would further benefit the short-term immunoprophylactic direct-acting Fab and Fc effector function mechanisms described above. Fc-enhanced antigen-binding proteins, such as the mAbs of the present invention, may provide more pronounced benefits by improving targeting of antigen-presenting cells and engagement of the host's adaptive immune response.

[0032] In another aspect, there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5; and a CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein has been modified to have an extended half-life.

[0033] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 11, a CDRH2 of SEQ ID NO: 12, a CDRH3 of SEQ ID NO: 13, a CDRL1 of SEQ ID NO: 14, a CDRL2 of SEQ ID NO: 15; and a CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein has been modified to have an extended half-life.

[0034] As used herein, the term extended half-life means that the time it takes for the serum concentration of an antigen-binding protein to reach half of its initial value is longer as measured in an FcRn binding assay, compared to a wild-type antigen-binding protein, or more particularly, an IgG1 antibody that does not contain a modification in the Fc region.

[0035] Several mechanisms for extending half-life are described throughout and are considered aspects of the invention described herein. In addition to amino acid substitutions that allow for improvements in ADNKA and ADCP, amino acid substitutions that allow for extended serum half-life were added because it is desirable to maintain serum concentrations of malaria mAbs for extended periods of time to minimize antibody administration in malaria-endemic countries that cover seasonal malaria.

[0036] In another embodiment, a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5; and a CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein is mutated at one of the following combinations of positions: M428L and N434S M252Y, S254T, and T256E H433K and N434F Further provided is the circumsporozoite protein (CSP) antigen binding protein, wherein the half-life of the antigen binding protein is extended.

[0037] In another embodiment, a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 11, a CDRH2 of SEQ ID NO: 12, a CDRH3 of SEQ ID NO: 13, a CDRL1 of SEQ ID NO: 14, a CDRL2 of SEQ ID NO: 15; and a CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein is mutated at one of the following combinations of positions: M428L and N434S M252Y, S254T, and T256E H433K and N434F Further provided is the circumsporozoite protein (CSP) antigen binding protein, wherein the half-life of the antigen binding protein is extended.

[0038] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5; and a CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein has been modified to have improved effector function and extended half-life.

[0039] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 11, a CDRH2 of SEQ ID NO: 12, a CDRH3 of SEQ ID NO: 13, a CDRL1 of SEQ ID NO: 14, a CDRL2 of SEQ ID NO: 15; and a CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein has been modified to have improved effector function and extended half-life.

[0040] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5; and a CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein comprises the following mutations: G236A, A330L, I332E, M428L and N434S.

[0041] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 11, a CDRH2 of SEQ ID NO: 12, a CDRH3 of SEQ ID NO: 13, a CDRL1 of SEQ ID NO: 14, a CDRL2 of SEQ ID NO: 15; and a CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein comprises the following mutations: G236A, A330L, I332E, M428L and N434S.

[0042] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5; and a CDRL3 of SEQ ID NO: 6, wherein the antigen binding protein comprises the following mutations: S239D, I332E, G236A, T250V, A287F, M428L and N434S.

[0043] In another aspect there is provided a circumsporozoite protein (CSP) antigen binding protein comprising a CDRH1 of SEQ ID NO: 11, a CDRH2 of SEQ ID NO: 12, a CDRH3 of SEQ ID NO: 13, a CDRL1 of SEQ ID NO: 14, a CDRL2 of SEQ ID NO: 15; and a CDRL3 of SEQ ID NO: 16, wherein the antigen binding protein comprises the following mutations: S239D, I332E, G236A, T250V, A287F, M428L and N434S.

[0044] In another aspect, there is provided a circumsporozoite protein (CSP) antigen binding protein comprising the sequence of a heavy chain variable region of SEQ ID NO:7 and a light chain variable region of SEQ ID NO:8, wherein the antigen binding protein has improved effector function and an extended half-life.

[0045] In another embodiment, there is provided a circumsporozoite protein (CSP) antigen binding protein comprising the sequence of a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8, wherein the antigen binding protein is mutated at positions G236A, A330L, I332E, M428L and N434S. In one embodiment, the antigen binding protein has improved effector function and extended half-life.

[0046] In another embodiment, there is provided a circumsporozoite protein (CSP) antigen binding protein comprising the sequence of a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8, wherein the antigen binding protein is mutated at positions S239D, I332E, G236A, T250V, A287F, M428L and N434S. In one embodiment, the antigen binding protein has improved effector function and extended half-life.

[0047] In another aspect, there is provided a circumsporozoite protein (CSP) antigen binding protein comprising the sequence of a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 18, wherein the antigen binding protein has improved effector function and an extended half-life.

[0048] In another embodiment, there is provided a circumsporozoite protein (CSP) antigen binding protein comprising the sequence of a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 18, wherein the antigen binding protein is mutated at positions G236A, A330L, I332E, M428L and N434S. In one embodiment, the antigen binding protein has improved effector function and extended half-life.

[0049] In another embodiment there is provided a circumsporozoite protein (CSP) antigen binding protein comprising the heavy chain sequence of SEQ ID NO:9 and the light chain sequence of SEQ ID NO:10.

[0050] In certain embodiments, 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 is provided.

[0051] In certain embodiments, one or more expression vectors are provided that comprise 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.

[0052] In certain embodiments, a recombinant host cell is provided that comprises one or more nucleic acid sequences 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 that comprise 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.

[0053] In one aspect, there is provided a method for producing a CSP antigen binding protein of the invention described herein, which method comprises culturing a host cell under conditions suitable for expression of one or more of said nucleic acid sequences or one or more vectors of the invention described herein, thereby producing a polypeptide comprising the CSP antigen binding protein.

[0054] In certain embodiments, a pharmaceutical composition is provided comprising a CSP antigen binding protein of the invention described herein and a pharmaceutically acceptable excipient.

[0055] In one embodiment, a method is provided for preventing malaria in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CSP antigen binding protein of the invention as described herein, or a pharmaceutical composition comprising a CSP antigen binding protein of the invention as described herein.

[0056] In one embodiment, the CSP antigen binding proteins of the invention described herein, or pharmaceutical compositions comprising the CSP antigen binding proteins of the invention described herein, are for use in the prevention of malaria.

[0057] In one embodiment, there is provided the use of a CSP binding protein of the invention described herein, or a pharmaceutical composition comprising a CSP binding protein of the invention described herein, in the manufacture of a medicament for use in the prevention of malaria.

[0058] As used herein, the term CSP binding protein refers to antibodies and other protein constructs, such as domains, that have the ability to bind to Plasmodium falciparum circumsporozoite protein. The terms CSP binding protein and CSP antigen binding protein are used interchangeably herein.

[0059] The term antibody is used herein in the broadest sense to refer to molecules having 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., as well as modified forms of any of the above (for a review of alternative antibody formats, see Holliger and Hudson, 2005, Nature Biotechnology, 23(9):1126-1136).

[0060] The terms "whole antibody," "whole antibody," or "intact antibody," used interchangeably herein, refer to a heterotetrameric glycoprotein with a molecular weight of approximately 150,000 daltons. An intact antibody consists of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. This H2L2 structure folds to form three functional domains, including two antigen-binding fragments known as "Fab" fragments and an "Fc" crystallizable fragment. The Fab fragment consists of an amino-terminal variable domain, either the heavy chain variable region (VH) or the light chain variable region (VL), and carboxyl-terminal constant domains, CH1 (heavy chain) and CL (light chain). The Fc fragment consists of two domains formed by the dimerization of paired CH2 and CH3 regions. The Fc fragment can exert 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 distinct heavy chain amino acid sequences designated μ, α, γ, ε, and δ, respectively, and each heavy chain can pair with either a κ or a λ light chain. The majority of antibodies in serum belong to the IgG class, but human IgG exists in four isotypes (IgG1, IgG2, IgG3, and IgG4) whose sequences differ primarily in the hinge region.

[0061] In certain embodiments of the invention described herein, the CSP antigen binding protein is an antibody. In other embodiments, it is a monoclonal antibody. In other embodiments, it is an IgG1 antibody.

[0062] Fully human antibodies can be obtained using a variety of methods, including the use of yeast-based libraries or transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies. Yeast displaying human antibodies on their surface that bind to the antigen of interest can be selected by FACS (fluorescence-activated cell sorting)-based methods or by capture on beads using labeled antigen. Transgenic animals engineered to express human immunoglobulin genes can be immunized with the antigen of interest, and antigen-specific human antibodies can be isolated using B-cell sorting techniques. Human antibodies generated using these techniques can be characterized for desirable properties, such as affinity, developability, and selectivity.

[0063] Alternative antibody formats include alternative scaffolds, in which one or more CDRs of an antigen binding protein may be placed onto a suitable non-immunoglobulin protein scaffold or framework, such as an affibody, an 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.

[0064] An antigen-binding site refers to a site on an antigen-binding protein capable of specifically binding to an antigen, which may be a single variable domain or the paired VH / VL domains found in a standard antibody. Single-chain Fv (ScFv) domains can also provide an antigen-binding site.

[0065] Affinity, also called binding affinity, is the strength of binding at a single interaction site, i.e., the strength of binding of one molecule, such as an antigen-binding protein of the present invention, to another molecule, such as its target antigen, at a single binding site. The binding affinity of an antigen-binding protein to a 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, binding affinity can be measured using the SPR method described in Example 1.

[0066] In some embodiments, the CSP antigen binding protein binds with an affinity of at least 2.5 nM as measured by surface plasmon resonance (SPR).

[0067] In some embodiments, the equilibrium dissociation constant (KD) of the antigen-binding protein interaction is 3 nM or less. For example, the antigen-binding protein interaction is 2.5 nM or less, or for example, 2 nM or less, or for example, 1 nM or less, or for example, 100 pM or less, or for example, 70 pM or less. In some embodiments, the antigen-binding protein interaction is 45 pM or less. One skilled in the art will appreciate that the smaller the KD number, the stronger the binding. In some embodiments, the antigen-binding protein interaction is 10 pM to 3 nM, or for example, 1 nM to 2.5 nM.

[0068] The term "neutralizing" as used throughout the present specification means that the biological activity of CSP is reduced in vitro or in vivo in the presence of an antigen-binding protein described herein, compared to the activity of CSP in the absence of the antigen-binding protein. Neutralization may be due to one or more of the following: blocking CSP binding to its receptor, inhibiting CSP from activating its receptor, inhibiting proteolysis of CSP, downregulating CSP or its receptor, or affecting the ability of an anti-CSP binding protein to neutralize its effector function.

[0069] CDR is defined as the amino acid sequence of the complementarity determining region of antigen-binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable region of immunoglobulin. Therefore, CDR as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy chain CDRs and all light chain CDRs, or at least two CDRs.

[0070] Throughout this specification, amino acid residues in variable domain sequences and variable domain regions within a full-length antigen-binding sequence (e.g., an antibody heavy chain sequence or an antibody light chain sequence) are numbered according to the Kabat numbering convention. Similarly, the terms CDR, CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 used in the examples follow the Kabat numbering convention. For further details, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987).

[0071] Those skilled in the art will appreciate that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. CDR sequences also have alternative numbering conventions, as described, for example, in Chothia et al., 1989, Nature, 342: 877-883. The structure and protein folding of antigen-binding proteins may mean that other residues are considered part of the CDR sequences, and this will be understood by those skilled in the art.

[0072] Other numbering conventions for CDR sequences available to those skilled in the art include the AbM (University of Bath) system and the contact (University College London) system.

[0073] Table A below provides one definition for each CDR or binding unit, using the respective numbering conventions. In Table A, the Kabat numbering scheme is used to number the variable domain amino acid sequences. It should be noted that some of the CDR definitions may vary depending on the individual paper used.

[0074] [Table A]

[0075] The CDRs can be modified by at least one amino acid substitution, deletion or addition, yet the variant antigen binding protein substantially retains the biological properties of the unmodified protein.

[0076] It will be appreciated that each of CDRH1, H2, H3, L1, L2, and L3 can be modified alone or in combination with other CDRs in any permutation or combination. In one embodiment, a CDR is modified by substitution, deletion, or addition of no more than three amino acids, for example, one or two amino acids, for example, one amino acid. Typically, the modifications are substitutions, particularly conservative substitutions, such as those shown in Table B below.

[0077] [Table B]

[0078] For example, in a variant CDR, the adjacent residues that comprise that CDR as part of alternative definitions such as Kabat or Chothia may be substituted with conservative amino acid residues.

[0079] Antigen binding proteins comprising variant CDRs as described above may be referred to herein as functional CDR variants.

[0080] The numbering used for a mutation or combination of mutations is done according to standard antibody numbering and will be understood by one of skill in the art.

[0081] Neutralization can be determined or measured using one or more assays known to those of skill in the art or as described herein.

[0082] The percent identity or % identity between a query nucleic acid sequence and a subject nucleic acid sequence is the percentage identity calculated over the entire length of the query sequence using an appropriate 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, etc.) after pairwise global sequence alignment using an appropriate 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 represented by a nucleic acid sequence disclosed herein, particularly in one or more claims.

[0083] The percent identity or % identity between a query nucleic acid sequence and a subject nucleic acid sequence is the percentage identity calculated over the entire length of the query sequence using an appropriate 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, etc.) after pairwise global sequence alignment using an appropriate 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 represented herein by a nucleic acid sequence disclosed, in particular, in one or more claims.

[0084] The query sequence may be 100% identical to the subject sequence, or may contain up to a certain integer number of amino acid or nucleotide modifications relative to the subject sequence such that the percent 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 subject sequence. In the case of nucleic acid sequences, such modifications include deletion, substitution, or insertion of at least one nucleotide residue, which may occur at the 5'- or 3'-terminal position of the query sequence, or anywhere between such terminal positions, and which are either individually interspersed among nucleotide residues in the query sequence or interspersed in one or more contiguous groups within the query sequence. In the case of amino acid sequences, such modifications include deletion, substitution (including conservative and non-conservative substitution), or insertion of at least one amino acid residue, which may occur at the amino- or carboxy-terminal position of the query sequence, or anywhere between those terminal positions, and which are interspersed individually among amino acid residues in the query sequence, or in one or more contiguous groups within the query sequence.

[0085] In the case of antibody sequences, the percent identity can be determined over the entire length of the query sequence, including the CDRs. Alternatively, the percent identity can exclude one or more, or all, of the CDRs; for example, if all CDRs are 100% identical to the subject sequence, variations in percent identity will be in the remainder of the query sequence, e.g., framework sequences, while the CDR sequences are fixed and intact. Variant sequences substantially retain the biological properties of the unmodified protein.

[0086] The term effector function, as used herein, refers to one or more antibody-mediated actions, including antibody-dependent NK activation (ADNKA), antibody-dependent cellular cytotoxicity (ADCC), antibody-mediated complement activation, including complement-dependent cytotoxicity (CDC), antibody-dependent complement-mediated cytolysis (ADCML), and Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP) by effector cells, including macrophages and neutrophils.

[0087] In further embodiments, effector function as used throughout this specification is intended to refer to one or more of antibody-dependent NK activation (ADNKA), antibody-dependent cellular cytotoxicity (ADCC), or antibody-dependent cellular phagocytosis (ADCP). In yet other embodiments, the effector function is ADCC.

[0088] Interactions between the Fc region of an antigen-binding protein or antibody and various Fc receptors (FcRs), such as FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcRn, C1q, and type II Fc receptors, and complement factors are thought to mediate the effector functions of the antigen-binding protein or antibody. Important biological effects can be the result of effector functionality. Typically, binding of an antigen-binding protein or antibody to an antigen is required to enable mediation of effector functions, and not all antigen-binding proteins or antibodies mediate all effector functions.

[0089] Effector function can be assessed in a number of ways, including, for example, assessing ADCC effector function of antibodies coated on target cells mediated by natural killer (NK) cells via FcγRIIIa, or ADCP effector function mediated by monocytes / macrophages or neutrophils via FcγRIIa, or assessing CDC effector function of antibodies coated on target cells mediated by the complement cascade via C1q. For example, antigen binding proteins of the invention can be assessed for ADCC effector function 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.

[0090] Throughout this specification, amino acid residues of the Fc region in an antibody sequence or full-length antigen-binding protein sequence are numbered according to the EU index numbering convention.

[0091] 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 contain any of the following mutations:

[0092] Enhanced CDC: Fc engineering can be used to enhance complement-based effector functions. For example, (for IgG1), K326W / E333S;S267E / H268F / S324T and IgG1 / IgG3 cross-subclassification can increase C1q binding; single E345R and triple E345R / E430G / S440Y mutations result in preformed IgG hexamers (Diebolder et al., Science 2014; 343: 1260-1263; Wang et al., 2018, Protein Cell, 9(1): 63-73).

[0093] Enhanced ADCC: Fc engineering can be used to enhance ADCC. For example (for IgG1), F243L / R292P / Y300L / V305I / P396L; S239D / I332E; and S298A / E333A / K334A increase FcγRIIIa binding; S239D / I332E / A330L increase FcγRIIIa binding and decrease 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 opposite heavy chain enhances FcγRIIIa F158 (low affinity allele) and FcγRIIIa F158 (low affinity allele). It increases affinity for V158 (high affinity allele) but does not increase binding affinity for inhibitory FcγRIIb (Wang et al, 2018, Protein Cell, 9(1):63-7).

[0094] Enhanced ADCPs: Fc engineering can be used to enhance ADCPs. For example, (for 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 FcγRIIa binding and improves the FcγRIIa / FcγRIIb binding ratio (activation / inhibition ratio), enhancing phagocytosis of antibody-coated target cells by macrophages (Wang et al., 2018, Protein Cell, 9(1):63-7).

[0095] Glycosylation The antigen-binding proteins of the present invention may comprise a heavy chain constant region with an altered glycosylation profile such that the antigen-binding protein has an enhanced effector function, for example, enhanced ADCC, enhanced CDC, enhanced ADCP, or a combination thereof. Examples of suitable methods for producing antigen-binding proteins with an altered glycosylation profile are described in WO2003011878, WO2006014679, and EP1229125, all of which can be applied to the antigen-binding proteins of the present invention.

[0096] The absence of the innermost α1,6 fucose residue in the Fc glycan moiety on N297 of IgG1 antibodies increases their affinity for FcγRIIIA, and therefore defucosylated or hypofucosylated monoclonal antibodies may have improved therapeutic efficacy (Shields et al, J Biol Chem, 2002, 277(30): 26733-40; Monnet et al, 2014, mAbs, 6(2):422-436).

[0097] COMPLEGENT In one embodiment of the present invention, an antigen-binding protein comprising a chimeric heavy chain constant region is provided. In one embodiment, the antigen-binding protein comprises an IgG1 / IgG3 chimeric heavy chain constant region, such 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 derived from IgG3. In one such embodiment, the antigen-binding protein may comprise one CH2 domain derived from IgG3, or both CH2 domains may be derived 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, except for position 435, which is H (histidine).

[0098] In one embodiment, the antigen binding protein comprises an IgG1 CH1 domain and at least one CH2 domain derived from IgG3. In one embodiment, the chimeric antigen binding protein comprises an IgG1 CH1 domain and further comprises the following residues in the CH2 domain, which correspond to IgG3 residues: 274Q, 276K, 296F, 300F, and 339T. In one embodiment, the chimeric antigen binding protein also comprises 356E in the CH3 domain, which corresponds to IgG3 residues. In one embodiment, the antigen binding protein also comprises one or more of the following residues in the CH3 domain, which correspond to IgG3 residues: 358M, 384S, 392N, 397M, 422I, 435R, and 436F.

[0099] Such methods for producing antigen-binding proteins having chimeric heavy chain constant regions can be carried out, for example, using the COMPLEGENT technology system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kirin Co., Ltd. The COMPLEGENT system, as described in WO2007011041 and US20070148165, each incorporated herein by reference, comprises a recombinant host cell containing an expression vector, in which a nucleic acid sequence encoding a chimeric Fc region having both IgG1 and IgG3 Fc region amino acid residues is expressed to produce an antigen-binding protein with enhanced CDC activity; i.e., CDC activity is increased compared to an identical antigen-binding protein lacking such chimeric Fc region. In another embodiment, CDC activity can be increased by introducing sequence-specific mutations into the Fc region of the IgG chain. Those skilled in the art will recognize other suitable systems.

[0100] POTELLIGENT Such methods of producing antigen-binding proteins can be performed, for example, using the POTELLIGENT technology system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with increased ADCC activity compared to the same monoclonal antibodies 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 skill in the art will recognize other suitable systems.

[0101] In an embodiment of the invention, the antigen binding protein is produced in a host cell in which the FUT8 gene has been inactivated. In an embodiment of the invention, the antigen binding protein is produced in a - / - FUT8 host cell. In an embodiment of the invention, the antigen binding protein is defucosylated at Asn297 (IgG1).

[0102] It will be apparent to one skilled in the art that such modifications can be used alone as well as in combination with each other to further enhance effector function.

[0103] The half-life of an antigen-binding protein refers to the time required for the serum concentration of the antigen-binding protein to decrease to half of its initial value. The serum half-life of a protein can be measured by pharmacokinetic studies according to the method described by 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 measured periodically as a function of time, for example, from about 3 minutes to about 72 hours after injection. Other methods for pharmacokinetic analysis and measuring the half-life of a molecule will be familiar to those skilled in the art.

[0104] It has been reported that the long half-life of IgG antibodies depends on binding to FcRn. Therefore, substitutions that increase the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependency of the interaction with the target by manipulating the constant region have been widely 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 protein of the present invention can be changed by modifying the heavy chain constant domain or FcRn-binding domain therein.

[0105] In adult mammals, FcRn, also known as the neonatal Fc receptor, plays a key role in maintaining serum antibody levels by functioning as a protective receptor that binds to antibodies of the IgG isotype and rescues them from degradation. IgG molecules are endocytosed by endothelial cells, and upon binding to FcRn, they exit the cell and are recycled into the circulation. On the other hand, intracellular IgG molecules that are not bound to FcR are routed to the lysosomal pathway, where they are degraded.

[0106] FcRn is thought to be involved in both antibody clearance and transcytosis across tissues (see Junghans, 1997, Immunol Res, 16:29-57 and Ghetie and Ward, 2000, Annu Rev Immunol 18:739-766). Residues of human IgG1 determined to directly interact with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435. Mutations at any of these positions may increase the serum half-life and / or alter the effector properties of the antigen-binding proteins of the invention.

[0107] The antigen-binding proteins of the present invention may have amino acid modifications that increase the affinity of the constant domain or fragment thereof for FcRn. Increasing the half-life (i.e., serum half-life) of therapeutic and diagnostic IgG antibodies and other biologically active molecules has many advantages, including reducing the dosage and / or frequency of administration of these molecules. In one embodiment, the antigen-binding proteins of the present invention comprise all or a portion of an IgG constant domain (FcRn-binding portion) with one or more of the following amino acid modifications:

[0108] For example, for 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).

[0109] Half-life can also be extended by T250Q / M428L, V259I / V308F / M428L, N434A, and T307A / E380A / N434A mutations (according to IgG1 and Kabat numbering) (Monnet et al, 2014, Mabs, 6(2):422-436).

[0110] Half-life and FcRn binding can also be increased (for IgG1) by introducing H433K and N434F mutations (commonly referred to as HN or NHance mutations) (WO2006 / 130834).

[0111] WO00 / 42072 discloses polypeptides comprising variant Fc regions with altered FcRn binding affinity, which comprise amino acid modifications at any one or more of the following amino acid positions in the Fc region: 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 (EU index numbering).

[0112] WO02 / 060919 discloses modified IgGs comprising an IgG constant domain that comprises one or more amino acid modifications relative to the wild-type IgG constant domain, which have an increased half-life compared to the half-life of an IgG having the wild-type IgG constant domain, and the one or more amino acid modifications are at one or more of positions 251, 253, 255, 285-290, 308-314, 385-389, and 428-435.

[0113] Shields et al. (2001, J Biol Chem, 276:6591-6604) used alanine-scanning mutagenesis to alter residues in the Fc region of a human IgG1 antibody and then evaluated binding to human FcRn. Positions that effectively inhibited FcRn binding when changed to alanine included I253, S254, H435, and Y436. The reduction in binding was less pronounced at other positions, including E233-G236, R255, K288, L309, S415, and H433. Several amino acid positions showed improved FcRn binding when changed to alanine; notable among these were P238, T256, E272, V305, T307, Q311, D312, K317, D376, E380, E382, S424, and N434. Many other amino acid positions showed either a slight improvement in FcRn binding (D265, N286, V303, K360, Q362, and A378) or no change (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, S330, R301, N315, E318, K320, K322, S330, R301, N316, E318, K322, K324, S332, R301, N317, E319, K336, K338, S339, R302, N318, E319, K340, K342, S343, R303, N319, E324, K345, S346, R301, N326, E327, K348, K349, S350, R302, N327, E328, K351, K352, S353, R303, N328, E329, K360, K362, S363, R304, N329, E335, K364, K365, S366, R305, N329, E336, K367, K368, S370 24, K326, A327, P329, P331, E333, K334, T335, S337, K338, K340, Q342, R344, E345, Q345, Q347, R356, M358, T359, K360, N361, Y373, S375, S383, N384, Q386, E388, N389, N390, K392, L398, S400, D401, K414, R416, Q418, Q419, N421, V422, E430, T437, K439, S440, S442, S444, and K447).

[0114] The most significant effect in terms of improved FcRn binding was seen with the combination variants. At pH 6.0, the E380A / N434A variant showed a greater than 8-fold improvement in FcRn binding over native IgG1, compared to 2-fold for E380A and 3.5-fold for N434A. Addition of T307A to this resulted in a 12-fold improvement in binding over native IgG1. In one embodiment, the antigen binding protein of the invention comprises the E380A / N434A mutations and has increased binding to FcRn.

[0115] 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. Random mutagenesis of positions 251, 252, 254-256, 308, 309, 311, 312, 314, 385-387, 389, 428, 433, 434, and 436 was disclosed. Substitutions of residues located in a strip across the Fc-FcRn interface (M252, S254, T256, H433, N434, and Y436) significantly improved the stability of the IgG1-human FcRn complex, whereas substitutions of peripheral residues such as V308, L309, Q311, G385, Q386, P387, and N389 produced less improvement. The variant with the highest affinity for human FcRn, derived from the combination of M252Y / S254T / T256E (YTE) and H433K / N434F / Y436H mutations, exhibited a 57-fold increase in affinity relative to wild-type IgG1. The in vivo behavior of this mutant human IgG1 showed a nearly fourfold increase in serum half-life in cynomolgus monkeys compared to wild-type IgG1.

[0116] Thus, the present invention provides antigen-binding proteins 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 said modification is at least one amino acid modification of any of the following amino acids: 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, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 5, 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.

[0117] Furthermore, various papers have described ways to obtain bioactive molecules with altered half-lives, either by introducing an FcRn-binding polypeptide into the molecule (WO97 / 43316, US5869046, US5747035, WO96 / 32478 and WO91 / 14438), or by fusing the molecule with an antibody that has preserved FcRn-binding affinity but significantly reduced affinity for other Fc receptors (WO99 / 43713), or by fusing with the FcRn-binding domain of an antibody (WO00 / 09560, US4703039).

[0118] Screening at pH 6.0 identified Fc variants with enhanced FcRn affinity that improved both antibody cytotoxicity and half-life. Selected IgG variants can be engineered as hypofucosylated molecules. The resulting variants exhibited increased serum persistence and preserved enhanced ADCC in hFcRn mice (Monnet et al., 2014, mAbs, 6(2):422-436). Examples of variants include (according 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 / A3 30V / 382V / N389T / N434Y;T256N / A378V / S383N / N434Y;N315D / A330V / N361D / A38 7V / N434Y;V259I / N315D / M428L / N434Y;P230S / N315D / M428L / N434Y;F241L / V264 E / T307P / A378V / H433R; T250A / N389K / N434Y; V305A / N315D / A330V / P395A / N434Y; V264E / Q386R / P396L / N434S / K439R; E294del / T307P / N434Y (the "del" above indicates a deletion).

[0119] Substitutions within the constant region can significantly improve the function of IgG antibodies, but substitutions within strictly conserved constant regions carry the risk of immunogenicity in humans (De Groot and Martin, 2009, Clin Immunol 131: 189-201), while substitutions within highly diverse variable region sequences may be less immunogenic. Reports on variable regions include modifying CDR residues to improve binding affinity to antigens (Rothe et al., 2006, Expert Opin Biol Ther 6: 177-187; Bostrom et al., 2009, Methods Mol Biol 525: 353-376; Thie et al., Methods Mol Biol 525: 309-322, 2009), as well as to improve stability (Worn and Pluckthun, 2001, J Mol Biol 305: 989-1010; Ewert et al., 2004, Methods 34: 184-199) and to reduce immunogenicity risk (De Groot and Martin, 2009, Clin Immunol 131: 189-201; Jones et al., 2009, Methods Mol Biol 525: 405-423, xiv), including modifying CDR residues and framework residues. As reported, improved affinity for antigens can be achieved by affinity maturation using phage or ribosome display of randomized libraries.

[0120] Improved stability can be achieved through sequence- and structure-based rational design. Reduced immunogenicity risk (deimmunization) can be achieved through various humanization techniques and removal of potential T cell epitopes, which can be predicted using in silico techniques or in vitro assays. Furthermore, variable regions have been engineered to lower pIs. These antibodies have been observed to have longer half-lives compared to wild-type antibodies despite comparable FcRn binding. Engineering or selecting antibodies with pH-dependent antigen binding can alter the half-life of the antibody and / or antigen. For example, the half-life of an IgG2 antibody can be shortened if antigen-mediated clearance mechanisms normally degrade the antibody upon antigen binding. Similarly, antigen:antibody complexes can affect antigen half-life, either by extending it by protecting it from typical degradation processes or by shortening it through antibody-mediated degradation (target-mediated drug disposition). One embodiment relates to antibodies that have higher affinity for antigen at pH 7.4 compared to 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 greater than or equal to 2. For example, to improve the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the antibody, pH-sensitive binding can be imparted to the antibody by introducing histidine residues into the CDRs.

[0121] The antigen binding proteins described herein may be incorporated into pharmaceutical compositions for use in the prevention or treatment of the human diseases described herein, particularly the prevention of malaria. In one embodiment, the pharmaceutical composition comprises the antigen binding protein in combination with one or more pharmaceutically acceptable carriers and / or excipients.

[0122] In yet another embodiment, provided herein is a pharmaceutical composition for administering a CSP antigen binding protein of the invention to a mammalian subject, preferably a human, at risk of malaria in an amount and on a schedule sufficient to prevent Plasmodium infection. In another embodiment, the subject resides in a malaria-endemic area of ​​sub-Saharan Africa. In one such embodiment, the human subject is a child under the age of 5. In another embodiment, the subject is a pregnant woman.

[0123] Such compositions may comprise a CSP antigen binding protein 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 therapeutically effective amount refers to an amount sufficient to prevent, treat, or at least partially arrest malaria or symptoms of malaria. A therapeutically effective amount can be determined by monitoring a patient's response to treatment. Typical criteria for a therapeutically effective amount include an improvement or prevention of malaria symptoms in a patient, such as a reduction in parasite counts.

[0124] In some embodiments, prevention of malaria refers to the complete prevention of liver stage disease establishment and the complete elimination of blood stage disease.

[0125] In some embodiments, prevention of malaria is measured as the delay in time to onset of detection of the parasite in the blood of the subject, for example, children and pregnant women in malaria-endemic countries such as sub-Saharan Africa.

[0126] In one embodiment, the antigen binding protein of the present invention provides a reduced risk of malaria infection. The risk of infection is measured in clinical trials as the level of protection achieved across a patient population compared to placebo. For example, analysis can be based on the time to first Plasmodium falciparum infection over a 24-week period. The mean time to first infection in a patient population administered placebo is much faster than in a patient population administered mAb. In another example, the proportion of participants who are completely free of Plasmodium falciparum infection over a 24-week period compared to placebo is measured. In one embodiment, the calculated reduction in risk of infection following administration is at least about 20%, 30%, 40%, 50%, or at least 60%, such as 65%.

[0127] In some embodiments, the antigen binding proteins of the invention provide a reduced risk of developing clinical symptoms of malaria, hi some embodiments, the risk of clinical disease after receiving at least one dose, for example with a 3 month testing interval, may be reduced by at least about 20%, 30%, 40%, 50%, at least 60%, such as about 65%.

[0128] In one embodiment, the malaria is non-severe malaria. In another embodiment, the malaria is severe.

[0129] In one embodiment the antigen binding protein reduces the risk of developing asexual parasitemia of Plasmodium falciparum in a subject despite having a fever, for example above 37.5°C, or having had a fever within the last 24 hours.

[0130] In some embodiments, the reduced risk of malaria infection and the reduced risk of clinical manifestations of malaria are assessed over a period of three months after administration of the antigen binding protein.

[0131] In some embodiments, the CSP binding protein provides protection for at least three months after administration.

[0132] As used herein, clinical malaria is defined as the presence of malaria parasites in the blood. In yet another example, clinical malaria can be defined as a fever of 37.5°C or greater accompanied by asexual parasitemia of 500 or more Plasmodium falciparum parasites per μL of blood (e.g., sensitivity and specificity of 90% or greater).

[0133] The definition of severe malaria includes, for example, one or more of the following: severe malarial anemia (PCV<15%), cerebral malaria (Blantyre coma score<2), or severe disease of other body systems, which may include multiple seizures (two or more generalized convulsions in the past 24 hours), weakness (defined as inability to sit unaided), hypoglycemia (<2.2 mmol / dL or <40 mg / dL), clinically suspected acidosis, or circulatory collapse.

[0134] The amount effective for use will depend on factors such as age, weight, and route of administration. Single or multiple administrations of the antibody will depend on the dosage and frequency required and tolerated by the patient. In some embodiments, the antibody is administered at the pre-erythrocytic stage of infection, within a period that will prevent hepatocyte infection.

[0135] The amount effective for use will vary depending on the severity of the disease and the patient's overall health, including other factors such as age, weight, sex, and route of administration. Single or multiple administrations of the antibody will depend on the dosage and frequency required and tolerated by the patient. In some embodiments, the antibody is administered at the pre-erythrocytic stage of infection or within a period that will prevent or reduce hepatocyte infection.

[0136] In some embodiments, the antigen binding protein is administered at least once prior to the infectious disease outbreak period, in other embodiments, the antigen binding protein is administered at least twice prior to the infectious disease outbreak period, and in other embodiments, the antigen binding protein is administered at least three times.

[0137] Pharmaceutical compositions can be administered by injection or continuous infusion (for example, but not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, and intraportal). In certain embodiments, the composition is suitable for intramuscular or subcutaneous administration. In certain aspects, administration is intramuscular. In certain embodiments, administration is subcutaneous. Pharmaceutical compositions may also be suitable for topical administration (including, but not limited to, epicutaneous, inhaled, nasal, or ocular administration) or enteral administration (including, but not limited to, oral, vaginal, or rectal administration).

[0138] In some embodiments, the CSP antigen binding proteins of the invention described herein, or pharmaceutical compositions described herein, or pharmaceutical compositions comprising the CSP antigen binding proteins described herein, are for intravenous, subcutaneous, or intramuscular administration. The pharmaceutical composition may be included in a kit containing the antigen binding protein together with other pharmaceutical agents and / or instructions for use. For convenience, the kit may include predetermined amounts of reagents along with instructions for use. The kit may also include a device used to administer the pharmaceutical composition. In yet other embodiments, the kit may include a diagnostic agent for detecting infection.

[0139] As used herein, the terms individual, subject, and patient are used interchangeably. In one embodiment, the subject is an animal. In another embodiment, the subject is a mammal, such as a primate, e.g., a marmoset or monkey. In another embodiment, the subject is a human.

[0140] The antigen binding proteins described herein can also be used in methods of prevention or treatment. The antigen binding proteins described herein are used in an amount effective for prophylactic treatment. As used herein, a therapeutically effective dose or therapeutically effective amount refers to an amount sufficient to prevent, treat, or at least partially arrest malaria or the symptoms of malaria.

[0141] In one embodiment, the antigen binding proteins of the invention described herein are administered to a subject in need thereof, in another embodiment, the antigen binding proteins are administered to patients under the age of 5. In another embodiment, the antigen binding proteins of the invention described herein are administered to pregnant women.

[0142] In some embodiments 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.

[0143] Antigen binding proteins can be prepared by any of a number of conventional techniques, for example, they can be purified from cells that naturally express them (e.g., antibodies can be purified from the hybridomas that produce them), or they can be produced in recombinant expression systems.

[0144] A number of different expression systems and purification methods can be used to produce the antigen-binding proteins of the present invention. Generally, host cells are transformed with a recombinant expression vector encoding one or more desired antigen-binding proteins. Depending on the expression system, the expression vector may be maintained by the host as a separate genetic element or may be integrated into the host chromosome. A wide variety of host cells can be used, including, but not limited to, prokaryotes (such as gram-negative or gram-positive bacteria, e.g., Escherichia, Bacillus sp., Pseudomonas sp., Corynebacterium sp.); eukaryotes such as yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris) or fungi (e.g., Aspergillus sp.); or higher eukaryotes such as insect cells and mammalian-derived cell lines (e.g., CHO, NS0, PER.C6, HEK293, HeLa).

[0145] The host cell may be an isolated host cell. The host cell is not normally part of a multicellular organism (e.g., a plant or an animal). The host cell may be a non-human host cell.

[0146] Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are known in the art.

[0147] Cells can be cultured under conditions that promote expression of the antigen-binding protein using a variety of devices, including shaker flasks, spinner flasks, and bioreactors. The polypeptide is recovered by conventional protein purification procedures. Protein purification procedures typically consist of a series of unit operations consisting of various filtration and chromatography steps developed to selectively enrich and isolate the antigen-binding protein. The purified antigen-binding protein can be formulated into a pharmaceutically acceptable composition.

[0148] [Table C]

[0149] Throughout this specification, for the avoidance of doubt, the nomenclature 356-ALE-LS, AB-000356 ALE LS, and AB-356 ALE_LS are used interchangeably and refer to the same antibody. (Example) Example 1

[0150] In vitro and in vivo characterization of a hit panel of malaria mAbs A diverse set of 79 antibody sequences derived from volunteers protected in a phase 2 trial of the RTS,S vaccine were selected as the evaluation panel. The diversity of the hit antibody panel is shown in Figure 1.

[0151] Because antibody developability and manufacturability do not always align with in vitro potency and optimal efficacy using in vivo preclinical disease models, the hit panel was first evaluated using in vitro protein analysis assays to determine which antibodies should be further developed.

[0152] The remaining molecules in the hit panel were then subjected to small-scale (125 ml) antibody production, followed by protein analysis testing using size exclusion chromatography (SEC), mass spectrometry (MS), hydrophobic interaction chromatography (HIC), heparin-binding ELISA, and nano-differential scanning fluorimetry (nano-DSF) assays. Target binding affinity was examined using recombinant CSPs by surface plasmon resonance (SPR). Antibodies that did not meet defined criteria, such as those with lower than required purified 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), those that showed high retention times in HIC (Table 1), and those with poor onset melt profiles (Table 2), were removed from the process. Furthermore, SPR data indicated several antibodies that showed no measurable binding or low binding affinities >3.0 nM (Table 3).

[0153] Molecules that were not excluded (referred to as the lead panel) were then advanced to efficacy testing in the P. berghei mouse model of malaria sporozoite challenge.

[0154] [Table 1] TIFF2025527672000005.tif250161TIFF2025527672000006.tif55161

[0155] [Table 2] TIFF2025527672000008.tif249160TIFF2025527672000009.tif42160

[0156] [Table 3] TIFF2025527672000011.tif250159TIFF2025527672000012.tif56160 Example 2

[0157] In vivo efficacy evaluation of lead antibody panel The protective efficacy of the lead panel was evaluated using a Plasmodium berghei (P. berghei) mouse model of in vivo protection, which involves C57BL / 6 mice infected with a recombinant P. berghei strain carrying the P. falciparum CSP and also carrying a GFP transgene marker to allow visualization of sporozoite infection of hepatocytes (Lin et al., 2013, Methods Mol Biol, 923:507-22; Tewari et al., 2002, J Biol Chem, 277(49):47613-8).

[0158] Because we previously determined that 300 μg was the antibody dose that provided complete protection in this mouse model, we chose the 200 μg dose to better assess the hierarchy of antibody efficacy. Animals (n = 3) were intravenously administered each mAb 24 hours before challenge with 1,500 sporozoites. Animals were then examined for liver infection by bioluminescence 44 hours later, and for blood-stage disease by PCR on day 3 and by flow cytometry through day 13. The number of animals in each group that were completely protected was assessed as the primary endpoint of the study. A summary of the study results is shown in Table 4. The results showed a wide variability in protection levels, with eight mAbs providing complete protection in only one of three animals (33% protection), another eight mAbs providing complete protection in two of three animals (66% protection), while at the study endpoint of day 10, only two mAbs (mAbs 356 and 338) provided 100% complete protection. The other mAbs did not provide protection at the study endpoint. Interestingly, there is no clear relationship between antibody affinity and in vivo efficacy. The top 10 most effective molecules were further screened using a lower dose of 100 μg, and the data are summarized in Table 5. Antibodies 356 and 338 were again the most effective molecules.

[0159] [Table 4] TIFF2025527672000014.tif55163

[0160] [Table 5] Example 3

[0161] Selection of amino acid substitutions required to confer optimal Fc effector function. When incorporating various amino acid substitutions designed to confer increased Fc effector function and half-life into monoclonal antibodies, there is a risk that other properties of the desired antibody pharmaceutical profile, such as target binding affinity, in vivo efficacy, or manufacturability, may be compromised.

[0162] Therefore, various amino acid substitution variants were generated using the tool antibody (mAb A). These substitutions were incorporated along with the amino acid substitutions required to confer improved serum half-life. A table showing the Fc variants generated is provided (Table 6).

[0163] [Table 6]

[0164] Protein analytical developability profiling assays were performed, including: i) protein yield, heparin binding, serum stability, nano DSF, and HIC to assess the potential for efficient manufacturing; ii) human FcRn and CSP target binding to provide confidence that serum half-life and target binding were not compromised; and iii) human FcγR binding by SPR to assess the potential for enhanced effector function. Complement C1q binding ELISA studies were also performed to assess the potential for complement activation by each antibody variant. In addition to in vitro human FcγR binding studies, select variants were further evaluated for potential enhanced effector function using an in vitro functional ADNKA assay.

[0165] Yield data for 2.0 liter-scale Fc variant antibody production are shown in Table 7. Fc variants mAb-A-DEL-LS and mAb-A-DE-LS exhibit surprisingly low total protein yields (30 mg and 45 mg, respectively, from 2.0 liters of HEK culture) compared to other Fc variants, which generally yielded total protein yields exceeding 150 mg. By comparison, mAb-A, produced as a defucosylated molecule, had very low yields from 4 liters of CHO cell culture. Thus, for a given antibody, different amino acid substitutions have significantly different effects on protein yield. [Table 7]

[0166] Human FcγR binding data by SPR are shown in Table 8. The affinity data demonstrated a variety of distinct FcγR binding profiles, including two variants (mAb A-VLPLL-LS and mAb A-LPLL-LS) that displayed significantly different affinities between alleles of the same receptor. Across the variants, there were clear differences in binding affinity to FcγR IIIa, with mAb A-AAA-LS being the lowest and mAb A-DEL-LS being the highest. The FcγR IIa / IIb binding ratios also differed significantly. Across the FcγR panel, mAb-B-DE-LS displayed a very similar binding profile to mAb-A-DE-LS, indicating that specific sets of amino acid substitutions can result in similar improvements in binding affinity to human FcγRs in different antibodies.

[0167] [Table 8]

[0168] The nano-DSF data (Table 9) show a clear melting profile trend, with the DEA-, DEL-, and DEAL-containing variants all having Tm1 melting inflection points below 50°C, followed by a group of variants with Tm1 values ​​in the 51–60°C range, and three variants with Tm1 values ​​between 60 and 65°C. The mAb-A (wild-type) molecule has a Tm1 of 69.45°C. The Tm1 of mAb-B-DE-LS is 51.39°C, very similar to the measured Tm1 of mAb-A-DE-LS (51.13°C), indicating that a specific set of amino acid substitutions can have similar effects between different antibodies. The aggregation onset, Tag, is fairly consistent between mAb-A variant molecules at 72 / 73°C, likely reflecting the melting of the Fab in both cases, while the Tag of mAb-B is several degrees lower. The variability in the Tm1 melting profiles likely indicates reversible melting of the CH2 region.

[0169] [Table 9]

[0170] Literature has shown that heparin binding is strongly correlated 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. The heparin-binding ELISA data (Figure 3) clearly demonstrate that all Fc variants exhibit lower heparin binding than the positive control antibody but higher heparin binding than the negative control antibody. The Fc variants mAb A-DE-LS, mAb A-LS-afucose, and mAb A-LPL-LS exhibited the highest mean heparin binding in this assay.

[0171] Complement C1q ELISA assay data (Table 10) show highly variable binding profiles among the different Fc variants. Variants mAb A-IE, mAb A-AE, and mAb-A SD exhibit high C1q binding, while DE-containing variants, particularly mAb A-DEL-LS and mAb A-DEAL-LS, as well as mAb A-ALE-LS, generally exhibit reduced C1q binding, at or near background levels. Furthermore, different CSP antibodies, such as mAb A-LS and mAb B-LS, have distinct C1q binding profiles.

[0172] [Table 10]

[0173] Human FcRn binding by SPR (Table 11) indicates that the introduction of various amino acid substitutions to improve effector function does not alter binding to human FcRn, and is not expected to have a significant effect on pharmacokinetics in vivo.

[0174] [Table 11]

[0175] Target CSP binding affinity was not altered by the introduction of the Fc substitutions (Table 12), and similarly hydrophobic interaction chromatography (HIC) showed no significant differences between the Fc variants (Table 13).

[0176] The in vitro stability of a panel of anti-CSP monoclonal antibodies after incubation in human serum was measured over an 8-week period. Each antibody was added to a pooled stock of human serum (n = 12 donors) to a concentration of 120 μg / ml. Seven individual samples were prepared from each stock, and in each case, all but one sample (designated TO) was incubated at 37°C for the indicated time periods (1, 2, 3, 4, 5, or 8 weeks). The TO sample was immediately frozen on dry ice and then stored at -80°C until all subsequent samples were collected and frozen in the same manner.

[0177] The remaining functional antibody in each series of samples after incubation was quantified using an MSD (Meso Scale Discovery) immunoassay, using CSP-coated plates for capture and a Sulfo-Tag-conjugated anti-human IgG1 Fc mAb for detection. A standard curve was constructed (using unstressed, sample-matched material) against which test samples were interpolated to determine concentrations. Recovery from each sample was expressed as a percentage of the initial TO sample.

[0178] The human serum stability data show no differences between Fc variants of the same molecule (Figure 4), but there are detectable differences between different antibodies, for example, mAb A and mAb B molecules. [Table 12] [Table 13]

[0179] Based on the data above, nine Fc variants were selected that possess favorable physical properties and are predicted to have enhanced Fc function (based on FcγR binding affinity). These Fc variants were further narrowed down to three using data generated by an in vitro human antibody-dependent NK cell activation (ADNKA) assay (Table 14), in which recombinant CSP is coated onto a microtiter plate and incubated with antibody followed by incubation with NK cells from a human donor. NK activation markers are then measured by flow cytometry. These studies demonstrate that all Fc modifications tested, particularly those containing the "DE" mutation and the "LPLIL" variant, enhance ADNKA in the presence of recombinant CSP. DEA, FTDE, and ALE were selected from this panel, and the variants selected for future evaluation were confirmed to enhance the full range of Fc-dependent mechanisms, based on their binding affinity to human FcγRs as shown in Table 8, based on published examples. As a result of this study, and to address the low Tm1 temperature associated with the DEA variant, the T250V / A287F (V / F) mutation was added to the DEA variant for future studies. [Table 14] Example 4

[0180] Incorporating amino acid substitutions that confer optimal Fc effector function into the most effective lead antibody Selected amino acid substitutions from Example 3 were incorporated into the sequences of mAb 356 and mAb 338, the most effective antibodies identified in the in vivo studies in Example 2. Functional in vitro assays were performed to confirm that improved effector function could 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 an ADCP assay measuring phagocytosis of recombinant CSP-coated beads by the monocytic cell line THP-1, all molecules tested were more potent than the mAb A reference standard (including mAb A variants with the DE and FTDE Fc modifications). Two 356 variants, 356-ALE-LS and 356-DEA-FV-LS, both had EC50s below 1 pM, making them more potent than any other molecules tested (Table 15). In the ADNKA assay, all molecules with ALE and DEA-FV produced enhanced ADNKA compared to the LS-only molecule (Table 16). [Table 15a] [Table 15b]

[0181] [Table 16]

[0182] To assess the in vivo impact of improved effector function, antibodies 356-ALE-LS and 356-DEA-FV-LS were compared to 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. Serum concentrations of each mAb at 2, 24, 44, and 96 hours post-dose are also shown. EpoFix (100 μg dose) and mAb A-LS (25 μg and 100 μg doses) were used as positive and negative infection controls.

[0183] [Table 17]

[0184] Statistical analysis of the PK / PD relationship between 356-ALE-LS and 356-LS showed that the effect of Fc-enhanced mAb 356-ALE-LS was small, 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. These probabilities decreased to 21.06% (day 2), 61.76% (day 3), and 5.55% (day 5) for a 3-fold increase in protective efficacy compared to the 356-LS molecule.

[0185] This very small difference in efficacy between 356-ALE-LS and 356-LS observed in the P. berghei model is not consistent with the much larger difference seen in the in vitro functional Fc assay described in Example 4. Given the known differences between human and mouse FcγR activity and cellular distribution, further in vivo studies will use mice transfected with human FcγRs, as the murine C57BL / 6 mouse background may not reflect the level of Fc-enhanced antibody activity consistent with human mAbs. Example 5

[0186] Safety analysis of lead antibodies The four lead antibodies were subjected to a variety of assays and tests to ensure (in an in vitro setting) that the antibodies were safe to develop, as far as possible, with regard to, for example, immunogenicity, tissue cross-reactivity, etc. The results are summarized below:

[0187] i) Immunogenicity studies of the four lead molecules, 356-ALE-LS, 356-DEA-FV-LS, 338-LS, and 338-ALE-LS, were performed using human peripheral blood mononuclear cells (PBMCs) from 20 different donors as a source of CD4+ T cells and dendritic cells, and CD4+ T cell proliferation and IFNγ production as readouts. The results provided an overall assessment among the four molecules: 356-ALE-LS induced positive responses in 2 of 17 donors (12% response rate), 356-DEA-FV-LS induced positive responses in 5 of 17 donors (29% response rate), and both 338-ALE-LS and 338-LS induced positive responses in 3 of 16 donors (19% response rate). Based on the assay qualification results and cumulative data from in-house immunogenicity risk testing, the above results suggest that it is likely that these mAbs will exhibit comparable potential to induce an immune response.

[0188] 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, representing non-specific binding of the mAbs, was observed in any of the five human tissues.

[0189] iii) Human off-target binding assessment was performed using cell microarray technology (Retrogenix). A panel of full-length plasma membrane proteins, plasma membrane-bound and secreted proteins, and heterodimer sets (over 6,400 human proteins) was tested for binding with the four lead mAbs at 20 μg / ml. Specific interactions were observed between SPOCK1 protein and two mAbs, 356-ALE-LS and 356-DEA-FV-LS. mAbs 338-LS and 338-ALE-LS showed specific interactions with CNTNAP2. To further explore these results, additional SPR binding data were generated using recombinant SPOCK1 and CNTNAP2 proteins and the four mAbs. These SPR results confirmed that none of the antibodies exhibited binding beyond nonspecific binding to the protein surface at 300 nM.

[0190] iv) SCISSOR (Subcutaneous Administration Site Simulation Device) is an in vitro assay designed to verify the bioavailability of molecules after subcutaneous administration. The predicted clinical bioavailability of GSK4425689A by SCISSOR analysis was 51.90% (NCR2022N506638_00). For context, this value is comparable to the bioavailability of a few approved comparator mAbs, which range from 52-80% (Zhao L et al. 2013). Example 6

[0191] In vitro and in vivo evaluation of Fc-enhanced mAbs in mice transgenic for human Fcγ receptors While mouse models have been used to demonstrate improved efficacy of Fc-enhanced mAbs (Hiatt et al., 2014, PNAS 111; 5992-5997; DiLillo et al., 2014, Nat Med 20(2) 143-151), the translational value of wild-type mouse models is uncertain due to known differences in the cellular distribution of effector receptors between human and mouse Fcγ receptors. Therefore, transgenic mice expressing human Fcγ receptors have been developed (Smith et al., 2012, Proc Natl Acad Sci U S A., 109(16): 6181-6186) and have been used to better predict the translational value of mAbs in disease (Casey et al., 2018, Leukemia, 32(2):547-549).

[0192] Our previous studies have shown that the in vitro activity of Fc-enhanced molecules is improved by more than 20-fold, while the in vivo activity in C57BL / 6 mice is only slightly improved, which is a significant difference. Therefore, we will investigate the possibility that the P. berghei model in C57BL / 6 mice underestimates the translational efficacy of Fc-enhanced mAbs using transgenic mice with human Fcγ receptors.

[0193] In vitro studies will be conducted to validate the transgenic mice, specifically to demonstrate that NK cells isolated from transgenic mice, but not from C57BL / 6 mice, respond to activation in assays using antibodies bound to immobilized CSP. These data provide confidence that transgenic mice may 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 are less responsive to such Fc-enhanced mAbs.

[0194] Following the in vitro studies, in vivo studies using transgenic mice demonstrated that the Fc-enhanced mAb exhibited a higher range of efficacy compared to previous studies in P. berghei-infected C57BL / 6 mice, as shown in Figure 5 . Example 7

[0195] In vitro and in vivo evaluation of Fc-enhanced mAb in transgenic mice carrying human Fcγ receptors In vitro studies used to evaluate the activation potential of NK cells isolated from both wild-type C57BL / 6 mice and human Fcγ receptor-transgenic mice involved coating recombinant CSP onto microtiter plates, incubating with anti-CSP mAb, and then adding NK cells isolated from mouse splenocytes. The activation marker CD107a was measured by flow cytometry. The difference in response to mAb AB-000356 LS and AB-000356 ALE LS in human FcγR transgenic mice indicates that Fc-enhancing substitutions can enhance NK cell activation (Figure 6a), whereas studies using wild-type C57 / Bl6 mice (Figure 6b) show no NK activation signal above baseline. These in vitro studies may partially explain why C57BL / 6 mice respond poorly to Fc-enhanced mAbs.

[0196] Next, in vivo studies were performed using transgenic mice transfected with human Fcγ receptors. Briefly, two antibody doses (25 μg and 100 μg per mouse) were administered intravenously to both transgenic mice (n = 5 per group) and wild-type C57BL / 6 mice (n = 4 per group) 24 h before challenge with 1,500 sporozoites. Liver infection was assessed 44 h later, 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 356 ALE-LS. A negative control antibody (EpoFix) was also included. Blood levels of parasitemia on day 3 after sporozoite challenge are shown in Figure 7, and summary data are presented in Tables 18 and 19. The differences in protection levels between the different antibodies were not clear, and all antibodies provided comparable levels of protection compared to the negative control antibody. Pharmacokinetic data show that circulating antibody concentrations are similar between the molecules.

[0197] [Table 18]

[0198] [Table 19]

[0199] Although NK cells isolated from human transgenic mice were shown to respond strongly to Fc-enhanced 356-ALE-LS, no significant increase in protection was observed after sporozoite challenge. The reasons for this are unclear but may be due to a variety of factors, including differences between the mouse challenge model and humans, such as the presence and activity of specific effector cell subsets that are thought to be involved in human malaria disease protection but have not been evaluated in mice (Ty et al., 2023, Sci Transl Med,15(680):eadd901), the difference in the time period from infectious sporozoite challenge to the end of the liver stage of disease in mice compared with humans (approximately 7 days vs. 2 days, respectively), and the fact that the protocol used in these iv challenge studies does not reflect the natural route of infection via mosquito bite. Example 8

[0200] In vivo evaluation of antibodies in mice transplanted with human hepatocytes and infected with Plasmodium falciparum To evaluate the in vivo efficacy of 356-ALE-LS against the human malaria parasite P. falciparum, we used the human liver chimeric FRG huHep mouse model (Minkah et al., 2018, Front Immunol, 19(9):807). These mice were transplanted with human primary hepatocytes and are susceptible to P. falciparum sporozoite infection, supporting the development of full liver stages. Mice (n = 6 per group) were challenged by mosquito bite with 40 infected mosquitoes and received a single dose of 300 μg of 356-ALE-LS per mouse (Group 2). The antibody L9-LS (Group 3), which has shown high efficacy in the FRG model and recently completed a malaria challenge trial in humans, was also included in the study as a positive benchmark control. The EpoFix isotype antibody served as a negative control (Group 1). All six mice treated with 356-ALE-LS had no detectable parasitemia in peripheral blood 8 days after sporozoite challenge, comparable to the five mice in the L9-LS group that were completely protected (Fig. 8C). No differences in serum antibody concentrations were observed throughout the study (Fig. 8D). These data suggest that 356-ALE-LS is comparable to L9-LS, although the additional benefit of improved effector function is unclear due to the WT C57BL / 6 mouse background strain used.

[0201] 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 356-ALE-LS show binding to the target recombinant CSP with similar nM affinity.

[0202] [Table 20]

[0203] The binding affinity of 356 ALE-LS to human Fcγ receptors was measured by SPR. Binding affinity was also measured for 356 LS and the Fc-silenced variant 356 LAGA-LS. Two formats of human IgG1 antibody, WT and Fc-silenced (LAGA), were included as negative controls. The data are shown in Table 21. Compared to the parent 356-LS mAb, mAb 356-ALE-LS exhibits higher human FcγRIIa and FcγRIIIa binding affinity, as well as lower hFcγRIIb binding.

[0204] [Table 21]

[0205] The binding affinity of 356 ALE-LS to human FcRn was measured by SPR. Binding affinity was also measured for mAb-LS, as well as two controls: a human IgG1 WT antibody and antibody L9, which does not contain LS. The data are shown in Table 22. Both 356-ALE-LS and mAb A-LS showed the expected low binding affinity at pH 6.0.

[0206] [Table 22]

[0207] [Sequence table] CDRH1 of SEQ ID NO: 1:356 NFGMH SEQ ID NO: 2: CDRH2 356 VIWHDGSNKFYADSVEG SEQ ID NO: 3: CDRH3 DSLFYDHDNSGYYGY SEQ ID NO: 4: CDRL1 RASRSVTSKLA SEQ ID NO: 5: CDRL2 GASTRAT SEQ ID NO: 6: CDRL3 QQYNNGFT

[0208] SEQ ID NO: 7: Heavy chain variable region QVQLVESGGGVVQPGRSLRLSCAASGFTFRNFGMHWVRQTPGKGLEWVAVIWHDGSNKFYADSVEGRFTISRDNSKNMIYLQMNSLRVEDTAIYYCARDSLFYDHDNSGYYGYWGQGTLVTVSS

[0209] SEQ ID NO: 8: Light chain variable region QIVMTQSPATVSVSPGERATLSCRASRSVTSKLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNNGFTFGPGTKVDFK

[0210] SEQ ID NO: 9: Heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFRNFGMHWVRQTPGKGLEWVAVIWHHDGSNKFYADSVEGRFTISRDNSKNMIYLQMNSLRVEDTAIYYCARDSLFYDHDNSGYYGY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0211] SEQ ID NO: 10: Light chain QIVMTQSPATVSVSPGERATLSCRASRSVTSKLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNNGFTFGPGTKVDFK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 11 CDRH1 TYGMH SEQ ID NO: 12 CDRH2 IIWHDGSKEFYADSVKG SEQ ID NO: 13 CDRH3 DDFDSSGHSYFHY SEQ ID NO: 14 CDRL1 RSSQSLVHSDGNTYVH SEQ ID NO: 15 CDRL2 KVSNRDS SEQ ID NO: 16 CDRL3 MQGTQWWT

[0212] SEQ ID NO: 17 Heavy chain variable region QVQLVESGGGVVQPGRSLRLSCAASGFSFSTYGMHWIRQVPGKGLEWVAIIWHDGSKEFYADSVKGRFTISRDNSKKKLYLQMNSLRAEDTAIYYCVKDDFDSSGHSYFHYWGQGTLVTVSS

[0213] SEQ ID NO: 18 Light chain variable region GVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYVHWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTQWWTFGQGTKVEIK

[0214] SEQ ID NO: 19 heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFSFSTYGMHWIRQVPGKGLEWVAIIWHDGSKEFYADSVKGRFTISRDNSKKKLYLQMNSLRAEDTAIYYCVKDDFDSSGHSYFHYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0215] SEQ ID NO: 20 light chain GVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYVHWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTQWWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0216] SEQ ID NO: 21 Heavy chain variable region nucleotide sequence (356) CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCAGGCAGATCTCTGAGGCTGAGCTGTGCCGCCAGCGGCTTTACCTTCAGGAACTTCGGCATGCACTGGGTGAGGCAGACCCCCGGAAAAGGCCTGGAATGGGTGGCCGTGATTTGGCACGACGGCAGCAACAAATTCTACGCCGACAGCGTGGAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACATGATCTACCTGCAGATGAACAGCCTGAGGGTGGAGGACACCGCCATCTACTACTGCGCCAGGGACTCTCTGTTCTACGACCACGACAACTCTGGCTACTACGGCTACTGGGGACAGGGCACTCTGGTGACCGTGAGCAGC

[0217] Sequence number 22 light chain variable region nucleotide sequence (356) CAGATCGTGATGACCCAGAGCCCCGCCACCGTGAGCGTGAGCCCAGGAGAAAGGGCCACTCTGAGCTGCAGGGCAAGCAGGTCTGTGACCAGCAAGCTGGCATGGTACCAGCAGAAACCCGGCCAGGCTCCCAGGCTGCTGATCTATGGAGCCAGCACCAGGGCTACCGGCATTCCTGCCAGGTTTAGCGGAAGCGGCAGCGGCACCGAGTTCACCCTGACCATCTCTAGCCTGCAGAGCGAGGACTTCGCCGTGTACTTCTGCCAGCAGTACAACAACGGCTTCACCTTCGGCCCCGGCACCAAGGTGGACTTCAAG

[0218] Sequence number 2(原文可能有误,推测为23) heavy chain nucleotide sequence

[0219] Sequence number 24 light chain variable region nucleotide sequence (356) CAGATCGTGATGACCCAGAGCCCCGCCACCGTGAGCGTGAGCCCAGGAGAAAGGGCCACTCTGAGCTGCAGGGCAAGCAGGTCTGTGACCAGCAAGCTGGCATGGTACCAGCAGAAACCCGGCCAGGCTCCCAGGCTGCTGATCTATGGAGCCAGCACCAGGGCTACCGGCATTCCTGCCAGGTTTAGCGGAAGCGGCAGCGGCACCGAGTTCACCCTGACCATCTCTAGCCTGCAGAGCGAGGACTTCGCCGTGTACTTCTGCCAGCAGTACAACAACGGCTTCACCTTCGGCCCCGGCACCAAGGTGGACTTCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

[0220] Sequence number 25 heavy chain variable region nucleotide sequence (338) CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGAAGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCAGCTTCAGCACCTACGGCATGCATTGGATCAGGCAGGTGCCCGGCAAAGGCCTGGAATGGGTGGCCATCATCTGGCACGATGGCAGCAAGGAGTTCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAAGAAGCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCATCTACTACTGCGTCAAGGACGACTTCGACAGCAGCGGCCACAGCTACTTTCACTACTGGGGCCAGGGAACCCTGGTGACAGTGAGCAGC

[0221] SEQ ID NO: 26 Light chain variable region nucleotide sequence (338)<000.jpg>GGCGTGGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACACTGGGCCAGCCAGCAAGCATTAGCTGCAGGAGCTCTCAGAGCCTGGTGCACAGCGACGGCAACACCTACGTGCACTGGTTTCAGCAGAGGCCCGGCCAGTCTCCCAGGAGGCTGATCTACAAGGTGAGCAACAGGGATAGCGGCGTGCCCGATAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTTACCCTGAAGATCTCTAGGGTGGAGGCCGAGGACGTGGGCGTGTACTATTGCATGCAGGGCACCCAGTGGTGGACCTTCGGCCAGGGAACCAAGGTGGAGATCAAG

[0222] SEQ ID NO: 27 Heavy chain nucleotide sequence (338 ALE LS)

[0223] Sequence number 28 light chain nucleotide sequence (338 ALE LS) GGCGTGGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACACTGGGCCAGCCAGCAAGCATTAGCTGCAGGAGCTCTCAGAGCCTGGTGCACAGCGACGGCAACACCTACGTGCACTGGTTTCAGCAGAGGCCCGGCCAGTCTCCCAGGAGGCTGATCTACAAGGTGAGCAACAGGGATAGCGGCGTGCCCGATAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTTACCCTGAAGATCTCTAGGGTGGAGGCCGAGGACGTGGGCGTGTACTATTGCATGCAGGGCACCCAGTGGTGGACCTTCGGCCAGGGAACCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

[0224] Sequence number 29 356 DEA FV LS heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFRNFGMHWVRQTPGKGLEWVAVIWHHDGSNKFYADSVEGRFTISRDNSKNMIYLQMNSLRVEDTAIYYCARDSLFYDHDNSGYYGY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLAGPDVFLFPPKPKDVLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNFKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0225] SEQ ID NO: 30356 DEA FV LS light chain QIVMTQSPATVSVSPGERATLSCRASRSVTSKLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNNGFTFGPGTKVDFK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0226] SEQ ID NO: 31 356 Heavy chain nucleotide sequence

[0227] Array number 32 356 DEA FV heavy chain nucleotide sequence CAGATCGTGATGACCCAGAGCCCCGCCACCGTGAGCGTGAGCCCAGGAGAAAGGGCCACTCTGAGCTGCAGGGCAAGCAGGTCTGTGACCAGCAAGCTGGCATGGTACCAGCAGAAACCCGGCCAGGCTCCCAGGCTGCTGATCTATGGAGCCAGCACCAGGGCTACCGGCATTCCTGCCAGGTTTAGCGGAAGCGGCAGCGGCACCGAGTTCACCCTGACCATCTCTAGCCTGCAGAGCGAGGACTTCGCCGTGTACTTCTGCCAGCAGTACAACAACGGCTTCACCTTCGGCCCCGGCACCAAGGTGGACTTCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

Claims

1. The heavy chain (HC) sequence of SEQ ID NO: 9; and Light chain (LC) sequence of SEQ ID NO: 10 Plasmodium falciparum circumsporozoite protein (CSP) antigen-binding protein.

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

3. 3. The antigen binding protein of claim 1 or 2, wherein the CSP binding protein remains in circulation for 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. An expression vector 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. 6. A recombinant host cell comprising one or more nucleic acid sequences according to claim 4 or one or more expression vectors according to claim 5.

7. 10. A method for producing a CSP antigen binding protein, the method comprising culturing the host cell of claim 6 under conditions suitable for expression of said nucleic acid sequence(s) or vector(s), whereby a polypeptide comprising the CSP antigen binding protein is produced.

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

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

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

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

12. Use of a CSP binding protein according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 8 in the manufacture of a medicament for use in the prevention of malaria.

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